Antimicrobial compositions

ABSTRACT

A composition includes hesperdin and/or a Lamiaceae extract wherein a majority of the volatile components have been removed from the Lamiaceae extract. A method for applying the composition to a food such as meat, fish or poultry, including processed and fresh or unprocessed meat, poultry and fish is also provided.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. patent application Ser. No.14/615,527, filed Feb. 6, 2015, herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to compositions for extending color and/ormicrobial stability to food and in particular to compositions whichextend color and/or microbial stability to food, including, but notlimited to meat, fish or poultry (fresh/unprocessed and processed).These compositions that are effective in extending the color andmicrobiological stability of food.

BACKGROUND OF THE INVENTION

Food safety and prevention of food spoilage is an ever present concernworldwide, particularly within the meat industry. Spoilage of food is amajor economic problem for a food manufacturer. Food manufacturers needto protect the health and safety of the public by delivering productsthat are safe to eat. Such food must have a guaranteed shelf life,either at chilled or ambient temperature storage. Consumers prefer goodtasting food of high quality. This is difficult to achieve with chemicalpreservatives, harsh heating regimes and other processing measures. Foodsafety and protection is best achieved with a multiple preservationsystem using a combined approach of milder processing and naturalpreservatives. Foodborne micro-organisms are also less able to adapt andgrow in food preserved with different preservative measures.

There is much concern about food protection and the growth of foodspoilage organisms such as Listeria monocytogenes. This particularspecies is one of the most problematic spoilage microorganism in meat.The unusual physiological characteristics such as exceptional resistancetoward antimicrobials are largely responsible for their ability to causespoilage. Additionally, spoilage organisms can sometimes adapt todifferent preservatives and storage conditions, thus a combination ofpreservative measures can be more successful than individual measures.

There is an increasing need to develop economical, natural and effectivepreservative systems to meet the public demand for convenient, natural,safe, healthy, good quality products with guaranteed shelf life.Antimicrobial materials such as those derived from plants can be used aspreservatives in food to help meet this need. Such plant extracts areconsidered to be desirable because they are regarded as being natural.Moreover from a regulatory point of view, because of long term usage,plant extracts typically have GRAS (generally regarded as safe) status.There is also a continuing to desire to provide microbial protectionutilizing lower amounts of antimicrobial materials. Thus there is a needto provide new antimicrobial materials or new more effectivecombinations of antimicrobial materials.

Despite their natural origins, it is desirable that antimicrobialproducts from plants be used in the lowest possible amounts. This isdesirable not only for reasons of cost but also to meet consumer desireto minimize the amount of ‘additives’ in foodstuffs. Moreover, manyplant materials have an associated taste. Therefore in many demandingfood applications reduction of the amount of protectant from plantorigin is advantageous.

Meat manufacturers are looking for ways to enable them to supply retailoutlets from efficient, cost effective, central-processing centers.Increased shelf life with regard to spoilage (consumer safety) isrequired to make this possible as meat makes its way through longerdistribution channels from producer to retailer to consumer.

Color shelf life is important to consumer acceptance. Consumers judgethe freshness of meat by the presence of bright red oxymyoglobinpigment. Oxymyoglobin in fresh meat decreases with time during storageas it changes to the stable brown pigment, metmyoglobin. Althoughoxymyoglobin pigment fades during dark storage, for example in a meatlocker, pigment loss is most pronounced in lighted, refrigerated displaycases in retail establishments. Further, pigment loss is primarilycosmetic in nature, it has serious economic consequences. Consumers insearch of the freshest looking cuts avoid purchasing meat containingeven small amounts of brown metmyoglobin.

Shelf life associated with microbial spoilage is a serious issue. Thepotential liability associated with food borne illness outbreaks fromthe sale of microbe-contaminated meat is enormous. The meat industry andassociated retail outlets are seeking ways to insure consumers' safetyby preventing microbial contamination all along the manufacturingprocess. Process improvements such as carcass washing and carefullycontrolled low temperature processing are now routine in the industry.One method for increasing shelf life associated with microbial spoilageis to package the food, e.g. meat, using modified atmosphere package(MAP).

There is a need in the industry for antimicrobial methods and processeswhich are perceived by consumers as being more natural. Theantimicrobial activity of the composition comprising Lamiaceae extractand hesperidin has been the main subject of study. Most prior artindicate that the antimicrobial activity of the herbs is centered in thevolatile essential oil components.

P. M. Davidson and A. S. Naidu (in Natural Food Antimicrobial Systems,A. S. Naidu, ed., 2000, CRC Press, Boca Raton, pages 265-294) review theantimicrobial properties of phyto-phenolic compounds from essential oilsof spices, herbs, edible grains and seeds. The authors teach that theantimicrobial effects of spices and herbs are primarily due to thepresence of phenolic compounds in the essential oil fractions and thatsome monoterpenes seem to show some activity, as well. Carvacrol,p-cymene and thymol are identified as the major volatile components oforegano, thyme and savory that likely account for the observed activity.The active antimicrobial agents of rosemary have been suggested to beborneol, camphor, 1,8-cineole, alpha pinene, camphene, verbenone andbornyl acetate. The active constituent of sage has been suggested to bethujone. Minimum lethal concentrations of essential oils of thyme oilhave been shown to range from 225-900 ppm in cultures. Theseconcentrations of essential oils in foods would cause serious flavorproblems. Since culture experiments underestimate the concentrationnecessary for effectiveness in foods, the flavor problems in foods arelikely to be more serious than even the culture numbers suggest. Inanother portion of this reference, minimum inhibitory concentrations ofessential oils were stated as 1-2% for rosemary, 0.12-2% for thyme,0.12-2% for spearmint, 0.5-2% for sage, 0.5-2% for peppermint and0.12-2% for oregano. In the summary, the authors state thatconcentrations of antimicrobial compounds in herbs and spices are toolow to be used effectively without adverse effects on the sensorycharacteristics of a food.

Y. Kimura et al. in U.S. Pat. No. 4,380,506, teach a process forproducing a preservative having antioxidant and antimicrobial activity.The process involves partitioning an extract of herb spices betweenpolar and non-polar solvents. Some of the partitioned extracts showedantimicrobial activity against Gram positive Bacillus subtilismicroorganisms in culture media. The only taste criterion tested byKimura et al. was the bitterness. Kimura et al. remained silent as toessential oil taste perception. Kimura et al. did not deodorize theextract which means that the extract contained essential oils andimpacted the taste of the meat. This impact on taste teaches away fromusing rosemary extracts obtained by the process taught by Kimura et al.

D. Ninkov (WO 01/15680) teaches that pharmaceutical compositions can beprepared by combining extracts of essential oils from plants of theLamiaceae family with an organic acid. Ninkov teaches that theantimicrobial activity of the pharmaceutical composition is due to thepresence of organic phenols such as isopropyl o-cresol in the oilextract from the plant.

K. Shetty and R. G. Labbe, (Asia Pacific J. Clin. Nutr. (1998), 7(3/4),pages 270-276., describe work to clone Lamiacae plants to produceenhanced levels of essential oil components such as carvacrol andthymol. These essential oil components have some antimicrobialproperties but their commercial use is prevented by the strong flavorsimparted to foods by these volatile compounds.

J. Campo, M. Amiot and C. Nguyen-the (2000, Journal of Food Protection63, pages 1359-1368) teach that rosemary extract has antimicrobialproperties in culture studies. Minimum inhibitory concentrations variedwith the species of bacteria being tested, but ranged from 0.06-1%.These researchers suggest that rosemary extract may show promise infoods with low fat and low protein content, against Gram positiveorganisms. No food systems were actually studied in this reference. Thisreference did not study specifically Listeria.

E. Down, et al., “Comparison of Vitamin E, Natural Antioxidants andAntioxidant Combinations on the Lean Color and Retail Case-Life ofGround Beef Patties” published in October, 1999, describes the effect ofrosemary extract in combination with other natural antioxidants andvitamin E diet supplementation on the color life of non-MAP ground beef.This reference does not teach how to extend the microbial shelf life ofthe meat. The authors failed to demonstrate a red color improvement ofthe meat by using rosemary as the red color preservation in meat with anatural antioxidant containing rosemary could not statistically differfrom the control. The red color of the control alters withincommercially desirable period. The loss of as much of the red color inthe control as in meat with the rosemary from this reference teachesaway from using rosemary extract as stability agent capable ofpreserving the red color of the meat.

Ahn et al. “Effects of plant extracts on microbial growth, color change,and lipid oxidation in cooked beef”, Food Microbiol., Vol. 24, Issue 1,(2007): 7-14 show that rosemary extract, or rosemary oleoresin, forwhich the contents in phenolic diterpenes are not known, has anantilisterial effect. In this reference, grape seed extract and pinebark extract had a greater antilisterial effect than rosemary whichteaches away from using rosemary extract as the lead antilisterialnatural product in meat. Further, Ahn et al. 2007 have shown that theaddition of rosemary extract to meat significantly deteriorated the redcolor of the meat, as compared to the control that lost less of the redcolor or as compared to grape extract that significantly improved thepreservation of the red color of the meat. Therefore, Ahn et al. 2007teach one to not use rosemary extract as stability agent capable ofpreserving the red color of the meat.

United States Patent Application Publication No. 2004/131709 studiesshow that rosemary extract alone, Herbalox® Seasoning, in which themajority of the volatile oil components has been removed shows verylittle, if any, antimicrobial effect. This reference does not teach howto extend the Gram positive, more precisely antilisterial shelf life ofmeat.

In addition, plant derived antimicrobials from citrus reported in theprior art are acids not flavonoids. For example, prior patents directedto compounds from citrus essentially relate to acids. KR20040001441describes orange juice as a suppressor of germ growth. However, onlyless than approximately 1/50^(th) of the juice reported in KR20040001441could be used in meat without perceiving meat as sour. As meat takes uponly up to 7.2% of the solution rich in citric acid, final levels inhesperidin taken up in meat would then correspond to less than0.48%*1/50*7.2%=˜0.0007%. This reference does not teach whetherhesperidin could have an antilisterial effect in meat.

Lorente, José et al. “Chemical guide parameters for Spanish lemon(Citrus limon (L.) Burm.) juices.” Food chemistry 162 (2014): 186-191discloses that citrus juice has titratable acidity of 52.4 g/L, withcitric acid being the main component. According to Lorente et al. (2014)in such juice, hesperidin levels as compared to titratable acidity arelower by more than two orders of magnitude (257 to 484.8 mg/L), whichcorresponds to 0.26 to 0.48% hesperidin w/v. Adding such an acidiccomposition to meat would impact the meat taste already at low levels.

Aktas̨, Nesimi, and Mükerrem Kaya. “The influence of marinating with weakorganic acids and salts on the intramuscular connective tissue andsensory properties of beef.” European Food Research and Technology 213.2(2001): 88-94 show that adding a solution of from 1% weak acid(including citric acid) to meat confers to the meat the sour taste. Alsothey show that when marinated in proportions 1:1 w/v (meat/marinade) themeat gains in weight at most 7.2% following marinating in marinadescontaining citric acid.

In WO 2012/112337, it is reported that flavonoids, including hesperidin,may provide some active antimicrobial activity without informing on thenature of microbes, whether they are bacteria, whether they are Grampositive bacteria nor whether they are Listeria. WO 2012/112337 teachesthat active antimicrobial compounds are acids.

Moulehi, Ikram, et al. “Variety and ripening impact on phenoliccomposition and antioxidant activity of mandarin (Citrus reticulateBlanco) and bitter orange (Citrus aurantium L.) seeds extracts.”Industrial Crops and Products 39 (2012): 74-80 report that citrus seedextracts contain total flavonoids of 1.31 to 2.52 mg equivalentcatechins/g DW. As hesperidin represents <16% of total flavonoids ofcitrus seed extract, this means that hesperidin is present at ˜0.032% inDW citrus seed extract.

Mandalari, G., et al. “Antimicrobial activity of flavonoids extractedfrom bergamot (Citris bergamia Risso) peel, a byproduct of the essentialoil industry.” Journal of Applied Microbiology 103.6 (2007): 2056-2064disclose that in vitro, citrus extracts rich in flavonoids inhibit thegrowth of Gram negative bacteria only and have no effect on the growthof Gram positive bacteria, and have no effect on the growth of Listeria.Mandalari et al. (2007) show that neohesperidin in pure form has noeffect on Listerial growth in vitro.

Fernandez-Lopez, J., et al. “Antioxidant and antibacterial activities ofnatural extracts: application in beef meatballs.” Meat science 69.3(2005): 371-380 show that meat supplemented with citrus extractscontaining flavonoids, the main of which is hesperidin, has no effect onthe growth of Listeria monocytogenes. For example, Fernandez-Lopez etal. (2005) show that such extracts exert antimicrobial effects on otherbacterial strains, including Listeria innocua, but not on Listeriamonocytogenes.

Teachings of Mandalari et al. and Fernandez-Lopez et al. teach away fromusing hesperidin as antilisterial compound and do not render obvious touse any or combination of flavonoids from a citrus extract againstListeria monocytogenes in meat. For example, Mandalari et al. (2007)teach away from using hesperidin as antilisterial compound and do notrender obvious that a purified flavonoid could have an antilisterialeffect.

Punica extracts rich in ellagic acid have no antimicrobial effects inraw MAP meat. For example, Hayes et al. (Hayes, J. E., Stepanyan, V.,Allen, P., O'Grady, M. N., & Kerry, J. p. 2010). “Effect of lutein,sesamol, ellagic acid and olive leaf extract on the quality andshelf-life stability of packaged raw minced beef patties”, Meat science,84(4), 613-620.) (hereinafter “Hayes et al”) teach that ellagic acid(one of active compounds from the Punica extract) has no antimicrobialeffect on raw beef MAP meat stored in cold and when applied at 300 ppm.Hayes et al. teach that ellagic acid did not improve the preservation ofthe red color of raw beef MAP meat stored in cold and when applied at300 ppm or at 600 ppm. Hayes et al. teach away from using lowerconcentrations than 300 ppm in ellagic acid for antimicrobial effect.Hayes et al. teach away from using ellagic acid for improving the redcolor of meat.

The general problem of enhancing the shelf life of fresh meat withoutimpacting the taste, remains in preventing the growth of spoilageorganisms and pathogens and in preserving the red color of the meatthroughout the commercially desirable storage period.

SUMMARY OF THE INVENTION

According to one aspect in accordance with the present invention,compositions include a Lamiaceae (rosemary) extract comprising phenolicditerpenes wherein the extract is essentially free of the nativeessential oil; and/or hesperidin extract. Advantageously, the hesperidinextract is pure hesperidin, i.e. having a concentration of at least 80%.

Other aspects of the present method and composition are for use with afood which includes but is not limited to fresh meat, poultry and fish,and comprising the components of a composition of this disclosure, apackaged food product and a method of packaging food.

The present invention, in one form thereof, relates to a compositioncomprising hesperidin and/or a Lamiaceae extract wherein a majority ofthe volatile components have been removed from the Lamiaceae extract.

The present invention, in another form thereof relates to a food whichincludes but is not limited to meat, poultry and fish, including bothunprocessed or fresh meat, poultry and fish and processed meat, poultryand fish, containing a composition comprising hesperidin and a Lamiaceaextract wherein a majority of the volatile oil components have beenremoved from the Lamiaceae extract.

The present invention in another form thereof relates to a method forpackaging food. The method includes applying to or incorporating into afood which includes but is not limited to fresh or unprocessed meat,fish or poultry, as well as processed meat, fish and poultry, acomposition comprising hesperidin and a Lamiaceae extract wherein amajority of the volatile oil components have been removed from theLamiaceae extract. The method, optionally, further includes packagingthe food in an atmosphere that contains 20% or more oxygen. Inalternative further embodiments, the amount of oxygen may be as much as70% oxygen. In yet an alternative embodiment, a packaged food productcomprises a food, e.g. fresh/unprocessed meat, fish or poultry orprocessed meat, fish or poultry, packaged in a standard atmosphericenvironment.

The present invention in another form thereof relates to a method forpackaging food. The method includes applying to, or incorporating into afood which includes but is not limited to meat, fish or poultry(fresh/unprocessed and processed), a composition comprising hesperidinand a Lamiaceae extract wherein a majority of the volatile oilcomponents have been removed from the Lamiaceae extract and the food ispackaged in an environment which includes 20% or more oxygen.

In one form, the present invention is directed to the presence ofhesperidin having a concentration of at least 80% up to 99%, preference95% and added 56-5380 ppm in combination or not with rosemary extract tofood to inhibit the growth of Listerial monocytogenes.

The present invention in another form thereof relates to a food productcomprising a food and containing a composition comprising purehesperidin or Lamiaceae extract.

The present invention in another form thereof relates to a method forpackaging food comprising applying to or incorporating into a food, acomposition comprising pure hesperidin or Lamiaceae extract. Optionally,the further can further comprises packaging the food in an atmospherethat contains 20% or more oxygen.

The present invention in yet another form thereof relates to anantimicrobial composition comprising an effective amount of a phenolicditerpene and/or of hesperidin.

The present invention in another form thereof relates to anantibacterial composition comprising an effective amount of a phenolicditerpene and/or of hesperidin. In one advantageous further form, thecomposition is effective against Gram positive bacteria selected fromthe group consisting of Bacillus cereus, Staphylococcus aureus,Streptococcus mutans, Listeria monocytogenes, Clostridium perfringens,Enterococcus hirae and Mycobacterium bovis. In an alternative furtherform, the composition is effective against Gram negative bacteriaselected from the group consisting of Pseudomonas aeruginosa,Escherichia coli, Salmonella typhimirium and Enterobacter cloacae.

The present invention in yet another form thereof relates to anantifungal/anti-yeast composition comprising an effective amount of aphenolic diterpene and of hesperidin. In one advantageous form, thecomposition is effective against yeast including Saccharomycescerevisiae and Candida albicans.

Rosemary extract in accordance with the present invention improves thepreservation of the red color of meat and extends the microbial shelflife without impacting the meat flavor. The present inventors' studiesin actual meat systems use a deodorized rosemary extract: an extractfrom which the majority of the volatile essential oil components hasbeen removed and that does not impact the meat taste. When combined withhesperidin or with Punica extract, unexpectedly, synergisticantilisterial and synergistic color preservation effects are observed,without any impact on the food taste.

The present method and composition can provide ways for food suppliersof any food product or beverage, including meat manufacturers to provideretailers with products from cost-efficient, cost-effective centralprocessing centers. The present method and composition can extend theshelf-life of food, including fresh and processed meat, fish andpoultry, and provide food that has extended microbial and colorshelf-life in an atmosphere containing 70% or more oxygen and 30% ormore CO₂. The method, in accordance with this disclosure that usescombinations of extracts can be used to improve the preservation of themeat color, block Listerial growth in food, including meats, fish andpoultry (fresh/unprocessed and processed), and allow for the use oflower, but more effective, inhibitory concentrations of plant extracts,without negative flavor impacts.

The present method is particularly suited for use with modifiedatmosphere packaged (MAP) meats. MAP meats are packaged in gasimpermeable materials that maintain an atmosphere above the product.Mixtures of oxygen and carbon dioxide are often used in MAP meats.Mixtures of these gases work very well with the present method.

In sharp contrast to the present method and composition, the prior artteaches that citrus extracts containing hesperidin has no effect onListerial growth in vitro nor in a food including meat. Pure flavonoidssuch as hesperitin or neohesperidin do not have any effect on Listerialgrowth in vitro, nor in meat. Mandalari et al. (2007) disclose that invitro, citrus extracts rich in flavonoids inhibit the growth of Gramnegative bacteria only and have no effect on the growth of Gram positivebacteria, and have no effect on the growth of Listeria. Mandalari et al.(2007) show that neohesperidin in pure form has no effect on Listerialgrowth in vitro.

Fernandez-Lopez et al. (2005) show that meat supplemented with citrusextracts containing flavonoids, the main of which is hesperidin, has noeffect on the growth of Listeria monocytogenes. Fernandez-Lopez et al.(2005) show that such extracts exert antimicrobial effects on otherbacterial strains, including Listeria innocua, but not on Listeriamonocytogenes.

Teachings of Mandalari et al. and Fernandez-Lopez et al. teach away fromhesperidin as antilisterial compound and do not render obvious to useany or combination of flavonoids from a citrus extract against Listeriamonocytogenes in food. Further, the teachings of Mandalari et al. teachaway from hesperidin as antilisterial compound and do not render obviousthat a purified flavonoid could have an antilisterial effect.

The present discloses shows that when hesperidin is concentrated atleast 80% up to 99%, preference at 95%, it inhibits the growth ofListeria monocytogenes in meat. The prior art teaches or at the least,suggests that hesperidin does not have an effect on Listerial growth infood including meat. The present inventors found surprisingly, thathesperidin can be used in a food product/foodstuff, such as meat (e.g.minced meat) to extend its microbial, color and taste shelf life. Whenhesperidin alone is used, hesperidin is required to be used in higherconcentrations to ensure adequate antilisterial effects. Surprisinglyand unexpectedly, the addition of plant extracts comprising phenolicditerpenes to hesperidin, synergistically improves antilisterial effectsand allows the use of lower doses of each extract.

Also, surprisingly, hesperidin and Lamiaceae extracts have been found topreserve color in MAP ground beef in a synergistic manner. In samples ofground beef stored six (6) days in cold conditions, hesperidin plusrosemary extract exceeds the color preserving additive effect ofhesperidin or rosemary alone.

The prior art is replete with statements that hesperidin containingextracts have no inhibitory effect on Listeria monocytogenes organisms.Surprisingly, the present inventors found evidence that hesperidin inthe presence of high oxygen concentrations inhibit Listeriamonocytogenes, Gram positive organism. The combination of hesperidin andhigh oxygen atmosphere inhibits Listeria monocytogenes, Gram positiveorganism isolated as a major spoilage organism in ground beef. Even moresurprisingly, combinations of rosemary and hesperidin show synergisticinhibition of these Listeria monocytogenes, Gram positive organisms,under high oxygen atmospheres.

The combinations of Lamiaceae extract containing phenolic diterpenes andhesperidin, preserve the color of meat, fish and poultry(fresh/unprocessed and processed) in the presence of oxygen in asynergistic manner.

Hesperidin alone at certain concentrations does not preserve the colorlife of fresh red meat and results in an unacceptable organolepticfeature. The combination of rosemary extract and hesperidin actssynergistically to extend the color life of ground beef in cold storageconditions. The combination is not just additive, but is synergistic,because it exceeds the additive effect of hesperidin alone and rosemaryalone.

The addition of Lamiaceae extract comprising phenolic diterpenes tohesperidin, yields in flavor acceptable composition which is effectivein preserving color and in inhibiting the growth of microorganisms inmeat, fish and poultry (both fresh/unprocessed and processed).

In accordance with the present disclosure, hesperidin alone suppressesListerial growth in food, including meat. Surprisingly, combinations ofLamiaceae extract, preferably, rosemary extract, and hesperidin, aremore effective in suppressing Gram positive, preferably Listeriamonocytogenes, bacterial growth than either Lamiaceae extract orhesperidin, alone.

The combination of Lamiaceae extract comprising phenolic diterpenes andhesperidin in the presence of oxygen, does not impact the flavor ofground beef in a package after a commercially desirable storage period.Neither Lamiaceae extract containing phenolic diterpenes nor hesperidinalone, or oxygen alone, or a combination of two of these factors alonepreserves the color as well as the combination of the three at the endof a commercially desirable storage period, without impacting theflavor.

Surprisingly, the addition of plant extracts comprising phenolicditerpenes, to hesperidin, synergistically improves antilisterialeffects and allows the use of lower doses of each extract.

Also, surprisingly, hesperidin and Lamiaceae extracts have been found topreserve color in MAP ground beef in a synergistic manner. In samples ofground beef stored five (5) days in cold conditions, hesperidin plusrosemary extract exceeds the color preserving additive effect ofhesperidin or rosemary alone.

The inventors found, surprisingly, that Punica extract containingpunicalagins and ellagic acid can be used in minced meat to extend itsmicrobial, color and taste shelf life. When Punica extract containingpunicalagins and ellagic acid are used alone (i.e. without rosemaryextract), higher concentrations of Punica extract are required to insureadequate antilisterial effects. Surprisingly, the addition of rosemaryextracts, or extracts of other Lamiaceae synergistically improvesantilisterial effects and allows the use of lower amounts of the Punicaextract (e.g. punicalagins and ellagic acid extracts).

Also, surprisingly, Punica extract containing punicalagins and ellagicacid and Lamiaceae extracts have been found to preserve color in MAPground beef in a synergistic manner. In samples of ground beef storedfive (5) days in cold conditions, Punica extract containing punicalaginsand ellagic acid plus rosemary extract exceeds the color preservingadditive effect of Punica or rosemary alone. This synergistic effect wasobserved at different concentrations of each extract.

Surprisingly, the inventors found evidence that Punica extractcontaining punicalagins and ellagic acid in the presence of high oxygenconcentrations inhibits Listeria monocytogenes, Gram positive organism.The combination of Punica extract containing punicalagins and ellagicacid and high oxygen atmosphere inhibits Listeria monocytogenes, Grampositive organism isolated as a major spoilage organism in ground beef.Even more surprisingly, the combination of rosemary and Punica extractcontaining punicalagins and ellagic acid show synergistic inhibition ofthese Listeria monocytogenes, Gram positive organisms, under high oxygenatmospheres.

The combinations of Lamiaceae extract containing phenolic diterpenes andPunica extract containing punicalagins and ellagic acid, extend thecolor shelf life of meat, fish and poultry (fresh/unprocessed andprocessed) in the presence of oxygen in a synergistic manner. Criticalto this invention is the combination of rosemary extract or othereffective Lamiaceae extract and Punica extract containing punicalaginsand ellagic acid, and the presence of oxygen.

Punica extract containing punicalagins and ellagic acid alone at certainconcentrations decreases the color life of red meat (e.g. fresh meat)and results in an unacceptable organoleptic feature. The combination ofrosemary extract and Punica extract containing punicalagins and ellagicacid acts synergistically to extend the color life of ground beef incold storage conditions. The combination is not just additive, but issynergistic, because it exceeds the additive effect of Punica extractcontaining punicalagins and ellagic acid alone and rosemary alone.

The addition of Lamiaceae extract containing phenolic diterpenes toPunica extract containing punicalagins and ellagic acid, yields inflavor acceptable composition which is effective in preserving color andin inhibiting the growth of microorganisms in meat, fish and poultry(fresh/unprocessed and processed).

The combinations of Lamiaceae extract, preferably, rosemary extract, andPunica extract containing punicalagins and ellagic acid, are moreeffective in suppressing Gram positive, preferably Listeriamonocytogenes, bacterial growth than either Lamiaceae extract or Punicaextract containing punicalagins and ellagic acid, alone.

The combination of Lamiaceae extract containing phenolic diterpenes andPunica extract containing punicalagins and ellagic acid in the presenceof oxygen, does not impact the flavor of ground beef in a package aftera commercially desirable storage period. Neither Lamiaceae extractcontaining phenolic diterpenes nor Punica extract containingpunicalagins and ellagic acid alone, or oxygen alone, or a combinationof two of these factors alone preserves the color as well and in asynergistic manner as the combination, at least up to sixth (6^(th)) dayof storage, without impacting the flavor.

Surprisingly, the addition of rosemary extracts, or extracts of otherLamiaceae synergistically improves antilisterial effects and allows theuse of lower doses of each extract.

Also, surprisingly, Punica extract containing punicalagins and ellagicacid and Lamiaceae extracts have been found to preserve color in MAPground beef in a synergistic manner. In samples of ground beef storedfive (5) days in cold conditions, Punica extract containing punicalaginsand ellagic acid plus rosemary extract exceeds the color preservingadditive effect of hesperidin or rosemary alone.

Surprisingly, the inventors found evidence that Punica extractcontaining punicalagins and ellagic acid in the presence of high oxygenconcentrations inhibit Listeria monocytogenes, Gram positive organism.The combination of Punica extract containing punicalagins and ellagicacid and high oxygen atmosphere inhibits Listeria monocytogenes, Grampositive organism isolated as a major spoilage organism in ground beef.Even more surprisingly, the combination of rosemary and Punica extractcontaining punicalagins and ellagic acid show synergistic inhibition ofthese Listeria monocytogenes, Gram positive organisms, under high oxygenatmospheres.

The combinations of Lamiaceae extract containing phenolic diterpenes andPunica extract containing punicalagins and ellagic acid, preserve thecolor of meat, fish and poultry (fresh/unprocessed and processed) in thepresence of oxygen in a synergistic manner. Accordingly, advantageous tosome of the present methods and compositions of this disclosure is acombination of rosemary extract or other effective Lamiaceae extract andPunica extract containing punicalagins and ellagic acid, and thepresence of oxygen.

Punica extract containing punicalagins and ellagic acid alone at certainconcentrations decreases the color life of red meat (e.g. fresh meat)and results in an unacceptable organoleptic feature. The combination ofrosemary extract and Punica extract containing punicalagins and ellagicacid acts synergistically to extend the color life of ground beef incold storage conditions. The combination is not just additive, but issynergistic, because it exceeds the additive effect of Punica extractcontaining punicalagins and ellagic acid alone and rosemary alone.

The addition of Lamiaceae extract containing phenolic diterpenes toPunica extract containing punicalagins and ellagic acid, yields inflavor acceptable composition which is effective in preserving color andin inhibiting the growth of microorganisms in meat, fish and poultry(fresh/unprocessed and processed).

The combinations of Lamiaceae extract, preferably, rosemary extract, andPunica extract containing punicalagins and ellagic acid, are moreeffective in synergistic manner in suppressing Gram positive, preferablyListeria monocytogenes, bacterial growth than either Lamiaceae extractor Punica extract containing punicalagins and ellagic acid, alone.Neither Lamiaceae extract containing phenolic diterpenes nor Punicaextract containing punicalagins and ellagic acid alone, or oxygen alone,or a combination of two of these factors alone preserves the meatagainst Listeria monocytogenes as well as the combination of the three,within commercially desirable storage period, without impacting theflavor.

The combination of Lamiaceae extract containing phenolic diterpenes andPunica extract containing punicalagins and ellagic acid in the presenceof oxygen, does not impact the flavor of ground beef in a package aftera commercially desirable storage period. Neither Lamiaceae extractcontaining phenolic diterpenes nor Punica extract containingpunicalagins and ellagic acid alone, or oxygen alone, or a combinationof two of these factors alone preserves the color as well as thecombination of the three after five (5) days of cold storage period,without impacting the flavor.

To keep the number of additives within reasonable bounds with respect tomeat, fish or poultry, it is advantageous to use botanical extracts thatprovide the property of inhibiting the growth of Listeria monocytogenes,and, more particularly, it is advantageous to combine botanical extractsthat provide synergistic antilisterial effects and that preserve the redcolor of the meat without impacting the meat taste. Advantageously,formulations of different botanical extracts, in accordance with thisdisclosure, function synergistically to increase the total antilisterialactivity and to preserve the red color of the meat without impacting themeat taste, of the combined extracts, that are superior to the sum oftheir individual contributions.

The methods and compositions in accordance with the present disclosure,exhibit a synergistic effect, as introduced above and will be discussedin more details to follow. It is noted that in contrast to thesynergistic effect of the methods and compositions in accordance withthe present disclosure, when two compounds elicit the same overtresponse, regardless of the mechanism of action and the combined effectis the algebraic sum of their individual effects, the compounds are saidto exhibit summation (Levine et al., 1996). However, in synergism, thejoint effect of two compounds is greater than the algebraic sum of theirindividual effects.

The technique in Levine et al. (1996) has been used to evaluatebiological effects of compound combinations. Shown in FIG. 1 (identifiedas “Prior Art” and taken from Basic Principles of Pharmacology, TulaneUniversity), top graph, compound combination effects illustrate thatwhen two compounds with similar mechanisms are given together, theytypically produce additive effects. This is also referred to assummation. However, if the effect of two compounds exceeds the sum oftheir individual effects, this is an unexpected effect referred tosynergism.

By analogy, a synergistic response concerning half doses, as illustratedin the bottom graph of FIG. 1, occurs if the combination of half thedose of compound A and B produces a response greater than A or B alone.

Those skilled in the art of antimicrobial formulations for food matricessuch as meat, are aware that antimicrobial synergy in meat is notpredictable. Not a single synergy could be disclosed for differentcombinations between three natural botanical extracts. Gutierrez, J.,Barry-Ryan, C., & Bourke, P. (2008). “The antimicrobial efficacy ofplant essential oil combinations and interactions with foodingredients.” International journal of food microbiology, 124(1),91-97). Synergistic effects of combinations have rarely been disclosedfor combinations between synthetic and natural extracts (see e.g. WO2013/169231).

One advantage of some compositions and methods, in accordance with thisdisclosure, is achieved by a process which removes volatile compounds bydeodorization. The deodorization process removes volatile compoundsincluding borneol, camphor, 1,8-cineole, alpha pinene, camphene,verbenone and bornyl acetate.

An additional advantage, in accordance with some aspects of the presentmethods, systems and compositions of this disclosure, is achievedthrough a combination of hesperidin or Punica extract with lamiaceaeextract. Further, unlike the process described in U.S. Pat. No.4,380,506, methods, in accordance with this disclosure, do not requirethe partitioning process and the methods avoid the use of additionalprocessing expense.

An additional advantage in accordance with one aspect of the presentmethod and system, is a composition which reduces the concentration ofvolatile compounds to a low level so as to not impact the taste of afood product to which a composition is applied such as meat, thereby notaffecting the taste of the meat.

One additional advantage of some aspects of the present invention is thepresence of Punica extract with more than 60 full lower concentration inellagic acid (than previously reported by Hayes et al) resulting inanti-listerial effects when combined with rosemary extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is adapted from “Basic Principles of Pharmacology”, (TulaneUniversity), in which the top portion is “Summation: Compounds A and BProduce Equal Effects, And Their Affects Are Additive When Combined” andthe bottom portion is “Synergism: The Combination of Half the Dose ofCompound A and Compound B Produces a Response Greater Than A or BAlone.”

FIG. 2 is a graph showing Listeria monocytogenes growth in meat inaccordance with the present invention.

FIG. 3 is a graph showing antilisterial synergy effects of combinationsof hesperidin and rosemary extract in accordance with the presentinvention.

FIG. 4 is a graph showing antimicrobial surface response of differentcombinations of rosemary extract and hesperidin inhibiting anddecreasing Listeria monocytogenes growth, in accordance with the presentinvention.

FIG. 5 is a bar chart showing red color values of the meat in accordancewith the present invention with regard to combinations of rosemaryextract and hesperidin in accordance with the present invention.

FIG. 6 is a bar chart showing red color values of meat in accordancewith the present invention with regard to various combinations ofrosemary and Punica extract in accordance with the present invention.

FIG. 7 is a chart showing inhibition of Listeria monocytogenes growth byplant extracts in minced beef.

FIG. 8 is a graph showing inhibition of Listeria monocytogenes growth byplant extracts in minced beef at 8° C. at day 9.

FIG. 9 is a chart showing inhibition of Listeria monocytogenes growth byplant extracts in minced beef at 8° C. at day 6.

FIG. 10 is a chart showing inhibition of Listeria monocytogenes e byplant extracts in minced beef at 8° C. at day 9 in accordance with thepresent invention.

FIG. 11 is a graph showing in accordance with the present invention,antimicrobial surface response of different combinations of rosemary andPunica extract inhibiting and decreasing Listeria monocytogenes growth.

FIG. 12 is a graph showing Listeria monocytogenes in poultry sausages.

FIG. 13 is a graph showing inhibition of Listeria growth in poultrysausages by an extract combination of R/P in accordance with the presentinvention.

FIG. 14 is a graph showing inhibition Listeria growth in poultry sausageby extract combination of R/H.

FIG. 15 is a graph showing Listeria monocytogenes in pork sausage(control).

FIG. 16 is a graph showing inhibition of Listeria growth in porksausages by an extract combination of R/P.

FIG. 17 is a graph showing inhibition of Listeria growth in porksaugsage by an extract combination of R/H.

FIG. 18 is a graph showing growth of Listeria monocytogenes in smokedsalmon (control).

FIG. 19 is a graph showing inhibition of Listeria monocytogenes insmoked salmon on the 30^(th) day of growth.

FIG. 20 is a graph showing inhibition of Listeria monocytogenes insmoked salmon on the 30^(th) day of grown.

DETAILED DESCRIPTION OF THE INVENTION

Compositions in accordance with this disclosure include Lamiaceaeextract and hesperidin and methods for using compositions for extendingthe shelf life of food including meat, fish and poultry (bothfresh/unprocessed and processed) without impacting the taste.

The present invention in another form, includes compositions comprisingLamiaceae extract and Punica extract and methods for using thesecompositions for extending the shelf life of food including meat, fishand poultry (both fresh/unprocessed and processed) without impacting thetaste.

The present methods and compositions are based on a discovery thatrosemary extracts rich in phenolic diterpenes, alone, or in combinationwith hesperidin or with Punica extracts rich in ellagic acid and inpunicalagins, preserve the red color of the meat for commerciallysignificant period. The present inventors discovered that treating meatwith pure hesperidin, that is a flavonoid, extracted from citrus peelsand then purified, prevents the growth of Listeria monocytogenes inmeat. Lamiaceae extracts comprising phenolic diterpenes in combinationwith hesperidin or with Punica extract comprising ellagic acid andpunicalagins, synergistically provided novel solutions for suppressingthe growth of microorganisms for a commercially desirable period and forpreserving the red color of the meat without impacting the meat taste.Compositions of this invention have been found to inhibit the growth ofGram positive microorganisms. Compositions of this invention, have beenfound to inhibit the growth of Listeria. Compositions of this invention,have been found to inhibit the growth of Listeria monocytogenes.

Combinations comprising: plant extracts standardized in phenolicditerpenes carnosic acid and carnosol, and hesperidin, or plant extractsstandardized in phenolic diterpenes carnosic acid and carnosol, andplant extracts standardized in ellagic acid and punicalagins, that wouldhave been used to synergistically prevent Listeria monocytogenes growthin food including meat, fish and poultry (both fresh/unprocessed andprocessed), without impacting the food taste and that synergisticallyimprove the preservation of the food color (e.g. meat color), could notbe retrieved from the prior art.

None of the prior art on the antimicrobial use of the combination ofrosemary or other Lamiaceae extracts comprising phenolic diterpenes withhesperidin or with Punica extracts comprising punicalagins and ellagicacid, either anticipates or renders obvious the present methods andcompositions. The prior art focuses on the use of herb essential oils oron the use of organic acids, such as citric acid. The rosemary extractsused in the present disclosure are processed in a manner that makes themessentially free of the native essential oil and rich in phenolicditerpenes. The prior art neither anticipates nor renders obvious thesynergistic combination of Lamiaceae extracts rich in phenolicditerpenes and Punica extracts rich in punicalagins and ellagic acid.

The prior art neither anticipates nor renders obvious the synergisticcombination of Lamiaceae extracts rich in phenolic diterpenes andhesperidin. The prior art neither anticipates nor renders obvious thesurprisingly beneficial antimicrobial effect of the combination ofLamiaceae extracts comprising phenolic diterpenes with Punica extractscomprising punicalagins and ellagic acid, or with hesperidin, on Grampositive organisms: Listeria monocytogenes. The prior art neitheranticipates nor renders obvious the surprisingly beneficial colorpreservation effect of the combination of Lamiaceae extracts comprisingphenolic diterpenes with Punica extract comprising punicalagins andellagic acid, or with hesperidin.

The prior art neither anticipates nor renders obvious the absence of theimpact on food taste of the combination of Lamiaceae extracts comprisingphenolic diterpenes with Punica extract comprising punicalagins andellagic acid, or with hesperidin.

Other flavonoids that have the same effect including but are not limitedto: narigin, isocurametin, neohesperidin, hesperidin, poncirin,nebiletin, and tangeretin.

Definitions

The following are a list of definitions used throughout this disclosure:

“Effective amount” is the amount necessary in order to achieve aspecific effect, in accordance with what one of ordinary skill in theart would be readily able to determine through routine experimentation.For example, with regard to the present disclosure, an effective amountof a composition comprising hesperidin and a Lamiaceae extract to beapplied to a food product or foodstuff, e.g. meat, fish and poultry(both fresh/unprocessed and processed), to extend the longevity of thefood, e.g. red color to the fresh meat, fish and poultry, is an amountwhich is determined to provide the red color longevity based on knownparameters which include, but are not limited to the concentration ofhesperidin and a Lamiaceae extract, the volume and/or surface area ofthe meat, fish and poultry, and the atmospheric environment conditionsof the meat, fish and poultry. Similarly, the effective amounts ofrosemary/Punica to extend the longevity of red color to the meat, fishand poultry are determined in a similar way.

“Food”, “food product” and “foodstuff” mean products that people oranimals eat. The food, food product and foodstuff include, but are notlimited to fresh and/or unprocessed meat, fish and poultry and processedmeat, fish and poultry.

“Fresh meat, fish, and poultry” means meat fish and poultry, entirecarcasses, cut portions thereof, and ground portions thereof. Freshmeat, fish, and poultry includes both unprocessed meat, fish and poultryas well as meat, fish, and poultry that includes additives such aspolyphosphates, salt, water, flavors, broths, added proteins, sugar,starches and the like which are incorporated into the meat, fish orpoultry. It is important to distinguish fresh meat, fish or poultrywhich may contain these ingredients, from “processed” meat, fish andpoultry which includes cured meat, fish and poultry, which may containthe same ingredients, but also contain one or more of the following:erythorbates, erythorbic acid, ascorbates, ascorbic acid, nitrites,nitrates or cultures. Fresh meat, fish and poultry are to bedistinguished from, and as opposed to, and does not include cured meat,fish or poultry, known as processed meat, fish and poultry.

“Hesperidin” means a compound extracted from nature or synthesized.

“Lamiaceae extract” means extract from a plant of the Lamiaceae family,preferably rosemary, sage, oregano, thyme, mints, and the followinggenera: Salvia, Rosmarinus, Lepechinia, Oreganum, Thymus, Hyssopus andmixtures thereof. The most preferred is rosemary.

“Meat, fish and poultry” means both a) processed meat, fish and poultryand b) unprocessed meat, fish and poultry.

“Phenolic diterpenes” means carnosic acid, carnosol, methylcarnosate,and other phenolic diterpene derivatives (rosmanol, isorosmanol,11,12-di-O-methylisorosmanol, 12-O-methylcarnosic acid, rosmanol-9-ethylether, circimaritin, Methylated monooxidized product of carnosic acid,genkwanin, epirosmanol, epiisorosmanol, carnosic acid derivative,epirosmanol ethyl ether, cryptotanshinone) and mixtures thereof.

“Processed” such as “processed foodstuff” and “processed meat, fish andpoultry” are products resulting from the processing of food, such asmeat, fish or poultry or from the further processing of such processedproducts, so that the cut surface shows that the product no longer hasthe characteristics of fresh meat, fish or poultry. Processing means anyaction that substantially alters the initial product, including heating,smoking, curing, maturing, drying, marinating, extraction, extrusion ora combination of those processes. Processes include non-heat treated andheat-treated processes.

“Punica extract” means extract from a plant of the Punica genus,preferably Punica granatum and Punica protoPunica, and mixtures thereof.The most preferred is Punica granatum.

“Pure hesperidin extract” means a hesperidin extract that has aconcentration of at least 80% hesperidin.

“Unprocessed” (such as meat, fish and poultry) means not havingundergone any treatment resulting in a substantial change in theoriginal state of the foodstuffs (e.g. meat, fish and poultry). However,the foodstuffs may have been for example divided, parted, severed,boned, minced, skinned, pared, peeled, ground, cut, cleaned, trimmed,deep-frozen, frozen, chilled, milled or husked, packed or unpacked.Unprocessed foodstuff, including meat, fish and poultry includeuntreated raw meat, fish and poultry, as well as fresh meat, fish andpoultry that has been comminuted or minced, that has had foodstuffsseasons or additives added to it or that has undergone processinginsufficient to modify the internal muscle fiber of the meat, fish orpoultry and thus eliminate the characteristics of fresh meat, fish orpoultry.

In the development of the present method and composition, it wasdiscovered that hesperidin has an antilisterial effect in meat whenprepared within certain ranges of concentrations.

In the development of the present method and composition, it wasdiscovered that rosemary extract comprising phenolic diterpenes combinedwith hesperidin or with Punica extract has a superior effect onsuppressing the growth of Listeria monocytogenes in meat than whenextracts are applied alone.

In the development of the present method and composition, it wasdiscovered that certain mixtures of extracts of the rosemary combinedwith hesperidin or with Punica extract comprising punicalagins andellagic acid, provide a synergistic antilisterial effect when preparedwithin certain ranges of concentration ratios.

In the development of the present method and composition, it wasdiscovered that rosemary extract comprising phenolic diterpenes combinedwith hesperidin or with Punica extract has a superior effect onpreserving the red color in meat than when extracts are applied alone.

In the development of the present method and composition, it wasdiscovered that certain mixtures of extracts of the rosemary combinedwith hesperidin or with Punica extract comprising punicalagins andellagic acid, provide a synergistic red color preservation effect inmeat when prepared within certain ranges of concentration ratios.

Mixtures of Extracts Rich in Phenolic Diterpenes and Hesperidin orPunica Extract

Phenolic diterpenes such as carnosic acid or carnosol occur specificallyin Lamiaceae. To date, carnosic acid has been identified in only a fewspecies, all exclusive of the Lamiaceae. To the best of the inventors'knowledge, only seven out of seventy (70) genera of the Mentheae tribecontain carnosic acid: Salvia (Brieskorn and Dumling, 1969), Rosmarinus(Luis and Johnson, 2005), Lepechinia (Bruno et al., 1991), Oreganum(Hossain et al., 2010) and Thymus (Achour et al., 2012). It may bepresent in Hyssopus where one of its possible derivatives,rosmanol-9-ethyl ether (7), was identified (Djarmati et al., 1991).Carnosic acid also occurs as a minor compound in one genus of theOcimeae tribe, Ocimum (Jayasinghe et al., 2003). Brieskorn, C. H.,Dumling, H. J., 1969. Carnosolsaure, der wichtige antioxydativ wirksameInhaltsstoff des Rosmarin-und Salbeiblattes. Zeitschrift furLebensmittel-Untersuchung and Forschung 141, 10-16; Luis, J. C.,Johnson, C. B., 2005; Bruno, Maurizio, et al. “Abietane diterpenoidsfrom Lepechinia meyeni and Lepechinia hastata.” Phytochemistry 30.7(1991): 2339-2343; Hossain, Mohammad B., et al. “Characterization ofphenolic composition in Lamiaceae spices by LC-ESI-MS/MS.” Journal ofagricultural and food chemistry 58.19 (2010): 10576-10581; Achour, S.,Khelifi, E., Attia, Y., Ferjani, E., Noureddine Hellah A., 2012.Concentration of Antioxidant Polyphenols from Thymus capitatus extractsby Membrane Process Technology. Journal of food science 77, C703-C709;Djarmati, Z., Jankov, R. M., Schwirtlich, E., Djulinac, B., Djordejevic,A., 1991. High antioxidant activity of extracts obtained from sage bysupercritical CO₂ extracton. Journal of the American Oil ChemistsSociety 68, 731-734; Jayasinghe, C., Gotoh, N., Aoki, T., Wada, S.,2003. Phenolic composition and antioxidant activity of sweet basil(Ocimum basilicum L.). Journal of agricultural and food chemistry 51,4442-4449. Seasonal variations of rosmarinic and carnosic acids inrosemary extracts. Analysis of their in vitro antiradical activity.Spanish Journal of Agricultural Research 3, 106-112.

Here these phenolic diterpenes were extracted from rosemary with the aimof extracting and concentrating essentially phenolic diterpenes: 44-85%.Thus obtained extract was then deodorized in order to get rid ofessential oils and volatile compounds that impact the food taste.

Rosemary Extract

Rosemary (Rosmarinus officinalis) leaves can be extracted with varioussolvents and yield extracts that are rich in different compounds. Forinstance, aqueous extracts are rather abundant in rosmarinic acidwhereas extractions using organic solvents rather yield in extracts richin phenolic diterpenes such as carnosic acid and carnosol. The detailedprocedure to prepare the composition of Rosemary extract was describedin the U.S. Pat. No. 5,859,293 and WO 96/34534, both herein incorporatedby reference.

The rosemary leaf was extracted with acetone at room temperature. Afterthe extraction was completed, the acetone extract was filtered toseparate the solution from rosemary leaf and concentrated under reducedpressure to make concentrated native extract. At this time, theconcentrated extract can be dried directly in a vacuum oven under mildheat to make a powdered extract, which is a composition comprising about15%-30% carnosic acid and 1%-3% carnosol. Alternatively, to theconcentrated native extract, aqueous sodium carbonate (NaHCO₃) was addedto dissolve carnosic acid and other organic acids, while base insolublesubstances were precipitated out.

The solution was filtered to separate from solid, and the filtrate wasfurther concentrated under reduced pressure. Once finishingconcentration is achieved, phosphoric acid (H₃PO₄) was added and theacid insoluble substances (including carnosic acid, carnosol, andcarnosic derivatives) were precipitated from the concentrated solution.Charcoal active is used during the process to decolorize the rosemaryextract in solution before filtration. Through filtering, theprecipitated solid was subsequently separated from liquid and rinsedwith water to remove impurities.

Last, the solid was dried in a vacuum oven and then milled into powderto make a composition containing about 40-65% carnosic acid, 2-10%carnosol, and 2-10% 12-O-methylcarnosic acid. Here used extractcontained >48% carnosic acid+carnosol. A last step was done to deodorizethe rosemary extract. It corresponded to a subsequent extraction of theprevious solid with a mix of acetone/hexane. The purpose of this stepwas the elimination of fatty molecules and of volatile compounds. Thefiltrate was concentrated under reduced pressure and was directlyformulated on liquid carrier.

Within the present specification and claims, this extract standardizedin phenolic diterpenes carnosic acid and carnosol, will be referred toeither as rosemary, or rosemary extract or rosemary (powder) or rosemary(liquid).

Hesperidin Extraction

Dried immature fruits (citrus aurantium L.) were exposed to a vapor inorder to remove pectins prior to the extraction with water.Subsequently, sodium hydroxide and calcium hydroxide were added in thesolution to stabilize the pH value. Following the filtration step, anacidification of the filtrate was induced using HCl. Upon this step thehesperidin precipitates, the liquid solution is removed and theprecipitate is dried. The final product (pure hesperidin) contains 90%to 99% hesperidin, preferably more than 95% of hesperidin as measured byHPLC.

The obtained extracts contain essentially hesperidin (>80%) and areconsidered to be pure. Throughout this disclosure, this extractstandardized in hesperidin at >95%, will be referred to as hesperidin orhesperidin (powder) or hesperidin (liquid).

Punica Extraction

Pomegranate skin bitter (Punica granatum L.) was extracted withethanol/water. The extract was filtered, then concentrated. The extractwas mixed with a carrier, in this example with maltodextrin prior todrying. Different drying technologies can be applied. This extract wasstandardized in following polyphenols: punicalagins (>7.5% by HPLC) andellagic acid (1.5-2.5%) as determined by HPLC.

Throughout this disclosure, this extract standardized in punicalagins(>7.5% by HPLC) and ellagic acid (1.5-2.5%), will be referred to eitheras Punica, or Punica extract or Punica (powder) or Punica (liquid).

Preparation of Products and Mixtures of Rosemary Extract/Hesperidin andRosemary Extract/Punica Extract

Plant extracts and their combinations were dried into powders.Maltodextrin was used in order to insure the suitable drying process ofcombinations of extracts. Maltodextrins are commonly used excipients orcarriers for drying processes.

Maltodextrins are defined as starch hydrolysis products with dextroseequivalent less than 20. Dextrose equivalent (DE value) is a measure ofthe reducing power of starch derived oligosaccharides expressed aspercentage of D-glucose on dry matter of hydrolysate and is inversevalue of average degree of polymerisation (DP) of anhydro glucose units.As products of starch hydrolysis, maltodextrins contain linear amyloseand branched amylopectin degradation products, therefore they areconsidered as D-glucose polymers joined by a-(1,4) and a-(1,6) linkages.

Although maltodextrins are derived from a natural compound (starch),their structure is different from the initial structure of the naturalmolecule they derive from (starch). This difference is induced by thehydrolysis process. Thus, maltodextrin structure does not occur innature.

Other possible excipients or carriers include maltodextrin, arabic gum,dextrose, salt, mono & diglycerides of fatty acids, MPG, Polysorbate 80,vegetable oil, mono & diglycerides of fatty acids, glucose syrup,glycerin, water and alcohol.

Compositions Were Added to the Raw Minced Beef Meat at 15% fat.

In the course of the work leading to the present method and composition,mixtures of rosemary extract and of hesperidin or of Punica extract, ina number of varying concentration ratios were tested for antilisterialeffectiveness using the classical microbiological methods. Bacterialenumeration in all here studied samples was performed on the Aloa mediumaccording to the standardized method (NF EN ISO 11-290). The growth ofListeria monocytogenes was evaluated in meat without any antilisterialagent and without any plant extract (control). The data of listerialgrowth in a control meat are represented in FIG. 2. It will be notedthat after 6 days of growth Listeria grew slightly, only by 0.29 logCFU/mL. After 9 days of growth, Listeria grew by 2.42 log CFU/mL.Experiments on meat were conducted in modified atmosphere packagings(MAP) that contained more than 20% O₂, more precisely 70% O₂ and 30%CO₂.

Following the meat manufacture, a batch of meat was sampled straightafter the mincing process and transported in refrigerated conditions tothe laboratory. In the laboratory, the meat was sampled into 2 kgsamples and conditioned in vacuum at −20° C., 24h prior toexperimentation, the 2 kg meat samples were transferred at 2-4° C. andkept at this temperature for 24 h±3 h until the core temperatureattained −1° C.

At this stage the 2 kg meat samples were inoculated with Listeriamonocytogenes in a laboratory of a biosafety of level 3 so that thecontamination by other microorganisms was avoided. Any furthersupplementation to the meat was conducted in such a laboratory.Following the homogenization of the inoculum at 4° C., the inoculated 2kg meat samples were supplemented with plant extracts and homogenized.Plant extracts were in powder form and were added as such to the meat.To keep them as dry powders, plant extracts were supplemented withmaltodextrin prior to drying process.

Plant extracts could be added as lipophilic or hydrophilic liquids, orcombinations thereof, to the meat. To do so, plant lipophilic orhydrophilic extract need to be solubilized or liquid, undried extractscould be used directly without undergoing the drying step.

Immediately after the supplementation of plant extracts andhomogenization, two pieces of 100 g of thus formed minced meat wereplaced together in trays. Control meat pieces, without extracttreatment, followed the same procedure.

Trays were then conditioned under modified atmosphere of 20% or more ofoxygen, preferably 70% O₂ and 30% CO₂ at 4 or at 8° C. Packaged meat wasstored in the dark for a stated amount of time.

A series of experiments involving rosemary and hesperidin extracts,rosemary and Punica extracts, typical antilisterial compounds (Sodiumlactate or Sodium acetate) and untreated control were conducted.Mixtures or alone extracts of rosemary and of hesperidin were added at1.18% to the meat. Mixtures or alone extracts of rosemary and of Punicawere added at 0.48% to the meat. Typical antilisterial compounds, Sodiumlactate and Sodium acetate, were added at classic concentrations 25 g/kgand 3 g/kg, respectively, in separate experiments.

Combinations of extracts were prepared and added to the meat accordingto the following proportions and doses prior to testing:

Control LM 0.5R R 0.5H H 0.5R + 0.5H 0.5R + H R + 0.5H R + H Compositionof Rosemary 0.00 1.28 2.56 0.00 0.00 1.28 1.28 2.56 2.56 extracts (%)extract Carnosic acid 0.00 0.56 1.13 0.00 0.00 0.56 0.56 1.13 1.13Carnosic acid + 0.00 0.62 1.24 0.00 0.00 0.62 0.62 1.24 1.24 carnosolHesperidin 0.00 0.00 0.00 24.00 49.00 24.00 48.00 24.00 48.00 extractHesperidin 0.00 0.00 0.00 22.80 46.55 22.80 45.60 22.80 45.60Composition in Rosemary 0 151 302 0 0 151 151 302 302 minced beefextract (ppm) Carnosic acid 0 66 133 0 0 66 66 133 133 Carnosic acid + 073 146 0 0 73 73 146 146 carnosol Hesperidin 0 0 0 2832 5782 2832 56642832 5664 extract Hesperidin 0 0 0 2690 5493 2690 5381 2690 5381 ControlLM 0.5R R 0.5P P 0.5R + 0.5P 0.5R + P R + 0.5P R + P CompositionRosemary extract 0.00 3.33 6.65 0.00 0.00 3.33 3.33 6.65 6.65 ofextracts (%) Carnosic acid 0.00 1.47 2.93 0.00 0.00 1.47 1.47 2.93 2.93Carnosic acid + 0.00 1.61 3.22 0.00 0.00 1.61 1.61 3.22 3.22 carnosolPomegranate extract 0.00 0.00 0.00 13.50 27.00 13.50 27.00 13.50 27.00Ellagic acid 0.00 0.00 0.00 0.27 0.54 0.27 0.54 0.27 0.54 Punicalagins0.00 0.00 0.00 1.22 2.43 1.22 2.43 1.22 2.43 Composition Rosemaryextract 0 160 319 0 0 160 160 319 319 in minced beef Carnosic acid 0 70140 0 0 70 70 140 140 (ppm) Carnosic acid + 0 77 154 0 0 77 77 154 154carnosol Pomegranate extract 0 0 0 648 1296 648 1296 648 1296 Ellagicacid 0 0 0 13 26 13 26 13 26 Punicalagins 0 0 0 58 117 58 117 58 117 R:rosemary extract; H: hesperidin extract; 0.5R: half concentration ofrosemary extract; 0.5H: half concentration of hesperidin extract R:rosemary extract; P: Punica extract; 0.5R: half concentration ofrosemary extract; 0.5P: half concentration of Punica extract

Immediately after the supplementation and the homogenization, two piecesof 100 g minced meat in shape of hamburgers were placed in trays. Thetrays were then conditioned in a modified atmosphere containing 70% O₂and 30% CO₂ and stored at 8° C. until analysis of Listerial growth andof organoleptic features, including the red color. Such analyses wereconducted on the 0^(th), 6^(th) and 9^(th) day of storage.

The growth of Listeria monocytogenes was evaluated in meat inrefrigerated conditions for each extract or compound and for theircombinations. The growth of Listeria monocytogenes was measured at thebeginning of the experiment, at ⅔rd of the commercial shelf life (6days) and at the time point corresponding to the commercial duration ofthe shelf life (9 days). Logarithmic values of Listerial growth (logCFU/mL) were calculated for each experiment and treatment. Differencesof logarithmic values of Listerial growth (log CFU/mL) between the meattreated with plant extracts and the untreated control were calculated toyield a final result. The more negative value was obtained, the higherwas the antilisterial effect of the extract or of the combination ofextracts. In meat science microbiology, for a given time, values areconsidered to be significant between two series when a difference of 0.5Log 10 CFU·g⁻¹ is observed (Chaillou et al., 2014); (Guide pour lavalidation de méthodes d'essais microbiologiques et l'évaluation de leurincertitude de mesure dans les domaines de la microbiologie alimentaireet de l'environnement), Schweizerische Eidgenossenschaft, Confédérationsuisse, Département féderal de l'économie, de la formation et de larecherche DEFR, Document No. 328, April 2013, Rev. 03). In microbiology,it will be noted that a treatment has a significant antibacterial effectif its effect exceeds −0.5 log CFU/mL as compared to the untreatedcontrol.

During the listerial growth, color of meat was monitored and images weretaken straight after the addition of the extract (on the day 0) and onthe 6^(th) day of growth). Images were taken under standardized lightconditions of exposure and using a system called “PackShot Creator.”Indeed, this professional equipment consists of an optimized light boxcontaining four fluorescent tubes diffusing homogeneous light, resultingin images always taken under the same conditions, with minimalreflection.

Each picture representing the “sample” at a different time scale wasloaded in the open source image analysis program ImageJ. The software iscommonly used in the food industry to measure different food parameterssuch as color or density (Reineke et al. “The Influence of Sugars onPressure Induced Starch Gelatinization, Procedia Food Science, 1, 2011,2040-3046; Kelkar et al. “Developing novel 3D measurement techniques andprediction method for food density determination , Procedia FoodScience, 1, 2011, 483-491). In order to obtain representative values ofthe red color, the color unit red (R) out of the three color units red(R) green (G) and blue (B) of the RGB model was used, and the color wasmeasured of each pixel of a line that was drawn across the sample. Theinbuilt RGB profile plot plugging was used to determine the differentcolor values of each pixel along this line, notably the values of thered color. The results are presented as variation of the different colorvalue as a function of the pixel number along this line. The resultswere statistically analyzed for significant differences using ANOVA testat p<0.05. Thus, per sample, more than 1000 pixels were analyzed.

In order to evaluate the effect of plant extracts on the color of themeat, red color of the treated meat was compared to an untreatedcontrol. The effect was calculated by [red color in meat withextract]−[red color in control meat (without extract)]. Negative effectmeans that the addition of extracts does not preserve the red color ofthe meat. Positive effect means that the addition of extracts improvesthe red color of the meat as compared to the control.

Mixtures of Rosemary and Hesperidin

Growth of Listeria monocytogenes in Raw Meat

Results of such testing at 6^(th) and 9^(th) day are presented in FIG. 7and FIG. 8. Data are means of 2 to 6 replicates. Data represent logdifferences in L. monocytogenes growth in treated meat as compared toinoculated controls (non-treated meat). Data were statistically analyzedfor significance at p<0.05 using ANOVA. Different letters indicatesignificant differences at p<0.05.

Results of such testing at 6^(th) day using rosemary extract and/orhesperidin are set forth in the following Table 1.

TABLE 1 Rosemary extract (%) Hesperidin (%) Expected effect Measuredeffect R 0 −0.08 0 H −0.09 R H −0.08 to −0.17 0.07* *Unexpected effectRosemary extract and/or hesperidin: full concentration effects onListeria monocytogenes growth after 6 days of growth in meat:[(log(CFU/mL) in meat treated with plant extracts) − (log(CFU/mL)control meat (without treatment))]

It will be noted that at such short duration (six (6) days of growth incold conditions), the difference in listerial growth in meat treatedwith plant extracts as compared with untreated meat, expressed in log,did not attain −0.5 log, which means that in such short time of growth,antilisterial effects could not be appreciated. It will be noted that atsuch short duration (six (6) days of growth in cold conditions),Listeria monocytogenes grew in control meat only by 0.29 log CFU/mL(FIG. 2).

It will be noted that when combined, the measured effect of thecombination of rosemary extract and of hesperidin does not correspond toa synergistic effect at the above concentrations after 6 days of growthas the combinatory effect is unexpectedly antagonistic.

When concentrations were halved, the following expected effectscalculated from the table above and measured effects were obtained andshown in Table 2.

TABLE 2 Rosemary extract (%) Hesperidin (%) Expected effect Measuredeffect 0.5R 0 −0.04 0.04* 0 0.5H −0.045 −0.37*   0.5R 0.5H −0.045 to−0.085 0.11* R 0.5H −0.045 to −0.125 −0.07   0.5R H −0.04 to −0.13 0*  *Unexpected effect Rosemary extract and hesperidin: half concentrationsand combinations of half and full concentrations effects on Listeriamonocytogenes growth after 6 days of growth in meat:[(log(CFU/mL) inmeat treated with plant extracts) − (log(CFU/mL) control meat (withouttreatment))]

It will be noted that at such short duration (6 days of growth in coldconditions), the difference in listerial growth in meat treated withplant extracts as compared with untreated meat, expressed in log, didnot attain −0.5 log, which means that in such short time of growth,antilisterial effects could not be appreciated. It will be noted that atsuch short duration (6 days of growth in cold conditions), Listeriamonocytogenes grew in control meat only by 0.29 log CFU/mL (FIG. 2).

It will be noted though, that, in the above Table 2, hesperidin appliedat a half dose alone has surprisingly a greater antilisterial effectthan at the full dose. Unexpected effect is signified by a star. On theother hand, effects of half dose of rosemary and of combination of halfdose rosemary and full dose hesperidin were antagonistic from what wasexpected. Finally, effects of combination of half dose rosemary and halfdose hesperidin and of combination of full dose rosemary and half dosehesperidin remained within additional range, as expected.

Results of such testing at 9^(th) day using rosemary extract and/orhesperidin are set forth in the following Table 3.

TABLE 3 Rosemary extract (%) Hesperidin (%) Expected effect Measuredeffect R 0 −1.12 0 H −0.64 R H −1.76 −0.85 *Unexpected effect Rosemaryextract and hesperidin: full concentration effects on Listeriamonocytogenes after 9 days of growth in meat: [(log(CFU/mL) in meattreated with plant extracts) − (log(CFU/mL) control meat (withouttreatment))]

It will be noted that after nine (9) days of growth in cold conditions,the difference in listerial growth expressed in log CFU/mL exceeded −0.5log CFU/mL, which means that antilisterial effects of all extracts andtheir concentrations and combinations presented in the above table couldbe appreciated within the commercially desirable period.

TABLE 4 Rosemary extract (%) Hesperidin (%) Expected effect Measuredeffect 0.5R 0 −0.56 −1.61* 0 0.5H −0.32 −0.71* 0.5R 0.5H −0.88 −1.64* R0.5H −1.83 −1.68 0.5R H −2.28 −0.82 *Unexpected effect Rosemary extractand hesperidin: half concentration and combinations of half and fullconcentrations effects on Listeria monocytogenes growth after 9 days ofgrowth in meat: [(log(CFU/mL) in meat treated with plant extracts) −(log(CFU/mL) control meat (without treatment))]

After nine (9) days of growth in meat, extracts alone or theircombinations at all tested concentrations inhibited the growth ofListeria monocytogenes by more than 0.5 log which means that they had anantilisterial effect in meat.

Unexpectedly in view of the prior art and in view of data in vitro,hesperidin had an antilisterial effect at all tested concentrations.Further, unexpectedly, rosemary extract or hesperidin alone had agreater antilisterial effect when used at half concentrations ascompared to full concentrations. Still further, unexpectedly, rosemaryextract combined with hesperidin at half concentrations had a greaterantilisterial effect than each extract alone at full concentration. Thisis synergy (FIG. 3).

Different concentrations and their response surfaces were analyzed usingsurface response methodology factorial experimental design that wasdesigned at three levels. These results are shown in FIG. 4. Theyindicate the following concentration ranges that provide antilisterialresponse in meat which is determined as: [(log(CFU/mL) in meat treatedwith plant extracts)-(log(CFU/mL) control meat (without treatment))]<0.5as provided in Table 5.

TABLE 5 Extract Extract proportion in combination (%) Hesperidin0.5-48.0 Rosemary extract 0.2-3.0  Extract proportions in combinationthat provide antilisterial response in meat (%)

It will be noted that to insure antilisterial effect, any of the aboveextract concentrations (Table 5) can be added in combination or alone tothe meat. The total percentage of the added extract, alone or incombination, to the meat did not exceed 1.18%.

During the listerial growth, color of meat was monitored and images weretaken straight after addition of the extract (on the day 0) and on the6^(th) day of growth).

Each picture representing the “sample” at a different time scale wasloaded in the open source image analysis program ImageJ. The software iscommonly used in the food industry to measure different food parameterssuch as color or density (Reineke et al. 2011; Kelkar et al. 2011. Inorder to obtain representative values of the three color units red (R),green (G) and blue (B) of the RGB models, a line was drawn across thesample. The inbuilt RGB profile plot plugin was used to determine thedifferent color values of each pixel along this line. The results arepresented as variation of the different color value as a function of thepixel number along this line. The results were statistically analyzedfor significant differences using ANOVA test at p<0.05. Per sample, morethan 1000 pixels were analyzed.

Red Color of the Raw Meat

The color of the meat was appreciated by a panel of sensorial analysis.This panel distinguished the meat color between bright red, red, brownand green hues. All meat samples were bright red on the day 0 ofexperiments.

On the 6^(th) day, the overall panel appreciation described the color ofdifferent meat samples subjected to different meat treatments asfollowing:

Meat color at Day 6 Control brown Sodium acetate brown Sodium lactatebrown 0.5R brown R red 0.5H green H brown 0.5R + 0.5H red 0.5R + H brownR + 0.5H red R + H brown

During the listerial growth, color of meat supplemented or not withplant extracts was monitored and images were taken straight afteraddition of the extract (on the day 0) and on the 6^(th) day of growth).

Results of such monitoring at 6^(th) day using rosemary extract andhesperidin alone or in combination are set forth in the following Table6:

TABLE 6 Rosemary extract (%) Hesperidin (%) Expected effect Measuredeffect R 0 11.05 0 H 10.69 R H 21.75 15.39

Rosemary extract and hesperidin of full concentration effects on redmeat color after 6 days of growth in meat. The effect was calculatedusing: [red color of meat with extract]−[red color of control meat(without extract)]

Contrary to the reports from the prior art, unexpectedly, Rosemaryextract better preserved the red color of the meat as compared to thecontrol. Hesperidin had slightly lower but similar effect.

The combination effect remains within the additional range and thereforewas not found to be synergistic at these concentrations.

The combination of extracts as compared to the control significantlyimproves the preservation of the red color more than each extract alone.

TABLE 7 Rosemary extract (%) Hesperidin (%) Expected effect Measuredeffect 0.5R 0 5.53 14.143* 0 0.5H 5.35 −1.63* 0.5R 0.5H 12.51 17.16* R0.5H 9.42 15.03* 0.5R H 16.22 13.33 *Unexpected effect Rosemary extractand hesperidin at full and half concentrations and combinations of halfand full concentrations effects on red meat color after 6 days of growthin meat. Each effect was calculated using: [red color of meat withextract] − [red color of control meat (without extract)]

FIG. 5 shows that when the concentration in hesperidin added to meat ishalved, it significantly decreased the preservation of the red color ofmeat as compared to the control.

Adding rosemary significantly improved the preservation of the meatcolor as compared to the untreated control meat. Unexpectedly, halvingrosemary concentration provoked a greater effect in red colorpreservation of meat than the full rosemary concentration. In addition,unexpectedly, halving hesperidin concentration did not yield in apreservation effect of the red color as expected but at thisconcentration, hesperidin deteriorated the preservation of the red coloras compared to the control. Further, unexpectedly, the effect on thepreservation of the red color of the meat of the combination of fullconcentration of rosemary and of half concentration of hesperidin,exceeded the expected additional effects of rosemary at fullconcentration or of hesperidin at halved concentration alone. This issynergy.

Still further, unexpectedly, the effect on the preservation of the redcolor of the meat of the combination of half a concentration of rosemaryand of half a concentration of hesperidin, exceeded the expectedadditional effects of half a concentration of rosemary or of hesperidinat halved concentration alone. This is synergy.

As to the rosemary extract at halved concentration and hesperidin atfull concentration, their combination effect remained within theadditional range and therefore was not found to be synergistic.

FIG. 5 shows that all combinations between rosemary and hesperidin atany here presented concentration significantly improved the preservationof the red color of the meat as compared to the control and as comparedto typical antilisterial compounds such as sodium acetate and sodiumlactate.

Mixtures of Rosemary and Punica

Growth of Listeria monocytogenes in Raw Meat

Results of such testing at 6^(th) and 9^(th) day are presented in FIG. 9and FIG. 10. Data are means of 2 to 6 replicates. Data represent logdifferences in L. monocytogenes growth in treated meat as compared toinoculated controls (non-treated meat). Data were statistically analyzedfor significance at p<0.05 using ANOVA. Different letters indicatesignificant differences at p<0.05.

TABLE 8 Rosemary Punica extract (%) extract (%) Expected effect Measuredeffect R 0 0.26 0 P 0.05 R P 0.31 0.12 *Unexpected effect Rosemaryextract and/or Punica extract: full concentration effects on Listeriamonocytogenes growth after 6 days of growth in meat: [(log(CFU/mL) inmeat treated with plant extracts) − (log(CFU/mL) control meat (withouttreatment))]

It will be noted that at such short duration (6 days of growth in coldconditions), the difference in listerial growth in meat treated withplant extracts as compared with untreated meat, expressed in log, didnot attain −0.5 log, which means that in such short time of growth,antilisterial effects could not be appreciated.

It will be noted that when combined, the measured effect of thecombination of rosemary extract and of Punica extract did not correspondto a synergistic effect.

When concentrations were halved, the following expected effectscalculated from the table above and measured effects were obtained:

TABLE 9 Rosemary Punica extract (%) extract (%) Expected effect Measuredeffect 0.5R 0 0.13 0.28 0 0.5P 0.025 0.27 0.5R 0.5P 0.55 0.11* R 0.5P0.53 −0.03* 0.5R P 0.33 0.26* *Unexpected effect Rosemary extract and/orPunica extract: half concentrations and combinations of half and fullconcentrations effects on Listeria monocytogenes growth after 6 days ofgrowth in meat: [(log(CFU/mL) in meat treated with plant extracts) −(log(CFU/mL) control meat (without treatment))]

It will be noted that at such short duration (6 days of growth in coldconditions), the difference in listerial growth expressed in log did notattain −0.5 log, which means that in such short time of growth,antilisterial effects could not be appreciated.

Unexpected Effect is Signified by a Star

Unexpectedly, the antilisterial effect of the combination of half aconcentration of rosemary and of half concentration of Punica, exceededthe expected additional effects of rosemary at halved concentration orof Punica at halved concentration, alone. This is synergy.

Further, unexpectedly, the antilisterial effect of the combination offull concentration of rosemary and of half concentration of Punica,exceeded the expected additional effects of rosemary at fullconcentration or of Punica at halved concentration, alone. This issynergy.

Still further, unexpectedly, the antilisterial effect of the combinationof half a concentration of rosemary and of full concentration of Punica,exceeded the expected additional effects of rosemary at halvedconcentration or of Punica at full concentration, alone. This issynergy.

Results of such testing at 9^(th) day using rosemary extract and/orPunica extract are set forth in Table 10.

TABLE 10 Rosemary Punica extract (%) extract (%) Expected effectMeasured effect R 0 −0.39 0 P −0.53 R P −0.92 −0.63 *Unexpected effectRosemary extract and Punica extract: full concentration effects onListeria monocytogenes growth after 9 days of growth in meat:[(log(CFU/mL) in meat treated with plant extracts) − (log(CFU/mL)control meat (without treatment))]

As mentioned above, in microbiology, it will be noted that a treatmenthas an antibacterial effect if its effect exceeds −0.5 log CFU/mL ascompared to the untreated control. It will be noted that after 9 days ofgrowth in cold conditions, compared to the control, the difference inlisterial growth expressed in log CFU/mL exceeded −0.5 log CFU/mL whenthe meat was treated with full concentrations of Punica or of thecombination of full concentration of rosemary and of full concentrationof Punica. Rosemary alone at the full concentration did notsignificantly inhibit the listerial growth as compared to the untreatedcontrol meat. However, combining rosemary at full concentration withPunica at full concentration had a greater antilisterial effect thanwhen extracts were used alone and enabled to exceed the threshold of−0.5 log CFU/mL that is required for a significant effect inantilisterial growth.

TABLE 11 Rosemary Punica extract (%) extract (%) Expected effectMeasured effect 0.5R 0 −0.195 −0.35* 0 0.5P −0.265 −0.84* 0.5R 0.5P−1.19 −1.47* R 0.5P −1.23 −1.45* 0.5R P −0.88 −0.69 *Unexpected effectRosemary extract and Punica extract: half concentrations andcombinations of half and full concentrations effects on Listeriamonocytogenes growth after 9 days of growth in meat: [(log(CFU/mL) inmeat treated with plant extracts) − (log(CFU/mL) control meat (withouttreatment))]

After 9 days of growth in meat, all but one extracts alone or theircombinations at almost all tested concentrations inhibited the growth ofListeria monocytogenes by more than 0.5 log CFU/mL as compared to thecontrol which means that they had an antilisterial effect in meat. Onlyrosemary extract alone when tested at half a concentration did notattain the difference of −0.5 log CFU/mL as compared to the control.

Unexpectedly, Punica extract alone had a greater antilisterial effectwhen used at half concentration as compared to a full concentration.Further, unexpectedly, when used at a half concentration, rosemaryextract had a greater antilisterial effect than expected. Still further,unexpectedly, the antilisterial effect of the combination of halfconcentration of rosemary and of half concentration of Punica extract,exceeded their expected additional effects of rosemary at halvedconcentration or of Punica extract at halved concentration alone. Thisis synergy.

In addition, unexpectedly, the antilisterial effect of the combinationof full concentration of rosemary and of half concentration of Punicaextract, exceeded the expected additional effects of rosemary at fullconcentration or of hesperidin at halved concentration, alone. This issynergy.

Further, unexpectedly, rosemary extract combined with Punica extract athalf concentrations had a greater antilisterial effect than each extractalone at full concentration. This is synergy (See e.g., FIG. 1).

Different concentrations and their response surfaces were analyzed usingsurface response methodology factorial experimental design that wasdesigned at three levels. These results are shown in FIG. 11. Theyindicate the following concentration ranges that provide antilisterialresponse in meat which is determined as [log(CFU/mL) in meat treatedwith plant extracts]−[log(CFU/mL) control meat (without plantextract)]<−1 as shown in Table 12.

TABLE 12 Extract Extract (%) Punica extract 5.0-24.0 Rosemary extract0.5-8.0  * Extract %

It will be noted that to insure antilisterial effect, any of the aboveextract concentrations (Table 12) can be added in combination or aloneto the meat. The total percentage of the added extract, alone or incombination, to the meat did not exceed 0.18%.

Red Color of the Raw Meat

The color of the meat was appreciated by a panel of sensorial analysis.This panel distinguished the meat color between bright red, red, brownand green hues. All meat samples were bright red on the day 0 ofexperiments.

On the 6^(th) day, the overall panel appreciation described the color ofdifferent meat samples subjected to different meat treatments asfollowing:

Meat color at Day 6 Control brown Sodium acetate brown Sodium lactatebrown 0.5R brown R brown 0.5P red P red 0.5R + 0.5P red 0.5R + P brownR + 0.5P red R + P brown

During the listerial growth, color of meat supplemented or not withplant extracts was monitored and images were taken straight afteraddition of the extract (on the day 0) and on the 6^(th) day of growth).Red pixels were quantified as explained above in the Methods section.

Results of such monitoring at 6^(th) day using rosemary extract andPunica extract alone or in combination are set forth in the following:

TABLE 13 Rosemary Punica extract (%) extract (%) Expected effectMeasured effect R 0 −3.72 0 P −5.60 R P −9.32 1.27* *Unexpected effectRosemary extract and Punica extract: full concentration effects on thepreservation of the red color of meat after 6 days of growth in meat.The effect was calculated by: [red color of meat with extract] − [redcolor of control meat (without extract)]

At the above concentrations (Table 13), when added alone, rosemary orPunica extract deteriorated the preservation of the red color of themeat as compared to the control. It was therefore expected that whencombined, these extracts would even further deteriorate the preservationof the red color of the meat. Unexpectedly, when combined, rosemary andPunica extracts improved the preservation of the red color of the meatas compared to the control.

Unexpectedly, the effect on the preservation of the red color of themeat of the combination of full concentration of rosemary and of fullconcentration of Punica, exceeded the expected additional effects ofrosemary at full concentration or of full concentration of Punica alone.This is synergy.

TABLE 14 Rosemary Punica extract (%) extract (%) Expected effectMeasured effect 0.5R 0 −1.86 1.07* 0 0.5P −2.80 −0.34* 0.5R 0.5P 0.734.45* R 0.5P −4.04 −2.71* 0.5R P −4.53 6.37* *Unexpected effect

Rosemary extract and Punica extract: full and half concentrations andcombinations of half and full concentrations effects on the preservationof the red color of the meat after 6 days of Listerial growth in meat.Each effect was calculated by: [red color of meat with extract]−[redcolor of control meat (without extract)]

As full concentrations of rosemary and of Punica extracts deterioratedthe preservation of the red color of the meat, it was expected thathalved concentrations would have also deteriorated the preservation ofthe red color of the meat. Unexpectedly, halving the added rosemaryconcentration significantly improved the preservation of the red colorof the meat as compared to the untreated control.

Adding rosemary significantly improved the preservation of the meatcolor as compared to the untreated control meat. Unexpectedly, halvingthe concentration of the added Punica extract did not deteriorate asmuch as expected the preservation of the red color of the meat.

Further, unexpectedly, the improvement of the preservation of the redcolor of the combination of half concentration of rosemary and of halfconcentration of Punica extract, exceeded their expected additionaleffects of rosemary at halved concentration or of Punica extract athalved concentration alone. This is synergy.

In addition, unexpectedly, the improvement of the preservation of thered color of the combination of full concentration of rosemary and ofhalf concentration of Punica extract, exceeded the expected additionaleffects of rosemary at full concentration or of hesperidin at halvedconcentration, alone. This is synergy.

Still further, unexpectedly, the improvement of the preservation of thered color of the combination of full concentration of Punica extract andof half concentration of rosemary extract, exceeded the expectedadditional effects of Punica extract at full concentration or ofrosemary extract at halved concentration, alone. This is synergy.

Unexpectedly, the combination of full concentration of Punica extractand of half concentration of rosemary extract improved the preservationof the color of the meat whereas it was expected that the preservationof the color be deteriorated upon application of such combination.

FIG. 6 shows that all combinations between rosemary and Punica extractat any here presented concentration significantly improved thepreservation of the red color of the meat as compared to the control andas compared to classic antilisterial agents such as Sodium acetate andSodium lactate.

One of ordinary skill in the art will recognize that additionalembodiments are also possible without departing from the teachings ofthe presently-disclosed subject matter. This detailed description, andparticularly the specific details of the exemplary embodiments disclosedherein, is given primarily for clarity of understanding, and nounnecessary limitations are to be understood therefrom, formodifications will become apparent to those skilled in the art uponreading this disclosure and can be made without departing from thespirit and scope of the presently-disclosed subject matter.

In vitro experiments:

Antimicrobial activities of Rosemary, Punica and hesperidin extractsalone and in combination Plant extracts of rosemary, hesperidin, Punicaand their combinations rosemary/hesperidin, rosemary/Punica wereprepared in 10% at DMSO_(50% final) according to the following protocolin which combinations of extracts were prepared, completed up to 100%with maltodextrin and added to the test solutions according to thefollowing proportions and doses prior to testing.

TABLE 15 R/H Composition of Rosemary extract 3.05 extracts (%) Carnosicacid 1.34 Carnosic acid + carnosol 1.48 Hesperidin extract 56.7Hesperidin 53.87 R: rosemary extract; H: hesperidin extract

TABLE 16 R P R/P Composition of Rosemary extract 5.33 0 5.33 extracts(%) Carnosic acid 2.35 0 2.35 Carnosic acid + carnosol 2.58 0 2.58Pomegranate extract 0 21.60 21.60 Ellagic acid 0 0.43 0.43 Punicalagins0 1.94 1.94 R: Rosemary extract; P: Punica extract

Preparation of the Working/Test Solution

200 mg of the extract (combination) were mixed with 1 ml 100% DMSO,vortexed, sonicated for 10 min at power 100%, 45 kHz, normal mode, wellvortexed, sonicated again and diluted 1:2 in sterile water at a finalconcentration of 100 mg/ml DMSO₅₀% final.

These preparations at 100 mg/ml were prepared in sterile 5 ml Eppendorftube. A sample was taken prior to preparing Minimum BactericidalConcentration (MBC) plates. MBC, Minimal Fungicidal Concentration (MFC)and Minimal Inhibitory Concentration (MIC).

Principle

The minimum bactericidal concentration (MBC) is the lowest sampleconcentration required to kill at least 99.99% of the inoculum (−4 log10). The minimum bactericidal concentration (MBC) is the lowestconcentration of an antibacterial agent required to kill a particularbacterium. It can be determined from broth dilution minimum inhibitoryconcentration (MIC) tests by subculturing to agar plates that do notcontain the test agent. The MBC is identified by determining the lowestconcentration of antibacterial agent that reduces the viability of theinitial bacterial inoculum by 99.9%. The MBC is complementary to theMIC; whereas the MIC test demonstrates the lowest level of antimicrobialagent that inhibits growth, the MBC demonstrates the lowest level ofantimicrobial agent that results in microbial death. This means thateven if a particular MIC shows inhibition, plating the bacteria ontoagar might still result in organism proliferation because theantimicrobial did not cause death. Antibacterial agents are usuallyregarded as bactericidal if the MBC is no more than four times the MIC.This test was based on the count of microorganisms in wells displayinglittle or no growth visually and then plated.

Samples prepared in 10% DMSO₅₀% _(final) were tested at concentrationsof 2.5, 0.5 and 0.1%, against one equivalent DMSO control (respectively12.5, 2.5 and 0.5%).

In the case of yeast, fungicide minimum concentrations (CMF) wereperformed as the WCD. In the case of the strain A. brasiliensis, afungus, the mere presence of growth was interpreted as absence offungicidal activity. The minimum inhibitory concentration (MIC) is thelowest concentration of an antimicrobial that will inhibit the visiblegrowth of a microorganism after adapted period of incubation. Minimuminhibitory concentrations are typically used to determine the potency ofnew antimicrobial agents, such as plant extracts or their combinations.The minimal inhibitory concentration or MIC is the lowest concentrationthat is sufficient to inhibit microbial growth by here tested extracts.This test is based on visual observation of the wells that containmicrobial strain that is studied with tested extracts or without(control). Time length and conditions of microbial growth were conductedaccording to classical methods well known by one ordinary skilled in theart.

The tests were performed on the following bacterial strains E. coli,Pseudomonas aeruginosa, Salmonella enterica ser typhimurium,Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes,Streptococcus mutans, Clostridium perfringens, Enterococcus hirae,Enterobacter cloacae, Moraxella bovis and two yeasts Saccharomycescerevisiae and Candida albicans and mold Aspergillus brasiliensis. Theevaluation of bactericidal and fungicidal activity was conductedaccording to the internal procedure in rich nutrient media , with acalibrated inoculum of 10⁵-10⁶ CFU/ml for bacteria and yeasts and 10⁵spores/ml for A. brasiliensis.

Prior to antimicrobial evaluation of extracts, the sterility of extractswas verified by plating. The absence of any microbial growth wascompulsory. Microbial cultures were conducted classically as summarizedin Table 17.

TABLE 17 Growth conditions and collections of microorganisms OrganismCollection Culture conditions Bacillus cereus ATCC 11.778 TS-30° C.-24 hStaphylococcus CIP 4.83 TS-37° C.-18 h aureus Listeria ATCC 19.115TS-37° C.-24 h monocytogenes Salmonella CIP 103.799 TS-37° C.-18 htyphimurium Escherichia coli CIP 53.126 TS-37° C.-18 h Pseudomonas CIP82.118 TS-37° C.-18 h aeruginosa Streptococcus ATCC 35.668 COS 5%sang/Broth Streptos- mutans 37° C.-24 h Enterococcus ATCC 8043 BrothTS-37° C.-24 h hirae Enterobacter CIP 103.475 Broth TS-30° C.-24 hcloacae Moraxella bovis CIP 70.40 T TS/Broth Col 10% horse serum-30°C.-24 h Clostridium ATCC 13.124 RCM/37° C./Anaerobiosis/48 h perfringensCandida albicans UMIP 48.72 Sabouraud/30° C./48 h Saccharomyces UMIP1181.79 Sabouraud/30° C./48 h cerevisiae Aspergillus niger IP 1431.83Sabouraud/30° C./48 h

Results

TABLE 18 MBC and MFC concentrations (0.1; 0.5 or 2.5%) of Rosemary (R),Punica (P), Rosemary/Punica (R/P) and Rosemary/Hesperidin (R/H) aseffective against the here presented microorganisms. R/H R/P S. aureus0.5 0.1 S. mutans 2.5 0.5 S. typhimirium 2.5 0.1 P. aeruginosa 2.5 0.1E. coli 2.5 0.5 L. monocytogenes 0.5 B. cereus 2.5 0.1 C. perfringens0.5 0.1 E. hirae 2.5 E. cloacae 0.5 M. bovis 0.1 0.1 R/H combination ofrosemary and hesperidin extracts according to Table 15; R: rosemaryextract, P: Punica extract, R/P: combination of rosemary and Punicaextracts according to Table 16. Empty cells: minimal concentrations (ifany) would be higher than the highest concentration here measured(2.5%).

TABLE 19 Unexpected/Synergistic MBC and MFC effects of R and P (% intest solution) Expected Measured MBC/MFC MBC/MFC MBC/MFC MBC/MFC of R(%) of P (%) of R/P (%) of R/P (%) S. typhimirium 0.5 0.5 0.25-0.5 0.1*P. aeruginosa 0.5 0.5 0.25-0.5 0.1* C. perfringens 0.03 >0.016 >0.0160.0078* *Unexpected result Unexpectedly, when applied in combination,the minimal concentration of the combination R/P to decrease microbialgrowth by 4 log, S. typhimirium and P. aeruginosa is surprisingly low;it is 2.5-5 times lower than expected. Surprisingly, when the twoextracts are combined, their activity is synergistically increased by2.5-5 times.

Unexpectedly, the anti-salmonella effect of the combination of rosemaryand Punica extracts, exceeded their expected additional effects whenextracts were applied alone. Indeed, it would have been expected that toachieve the same anti-salmonella effect and decrease microbial growth by4 log, MBC would have been at best halved when the extracts are combinedas compared to MBC when extracts were applied alone. Surprisingly, here,MBC were decreased by 2.5-5 times. This is synergy.

Further unexpectedly, the anti-pseudomonas effect of the combination ofrosemary and Punica extracts, exceeded their expected additional effectswhen extracts were applied alone. Indeed, it would have been expectedthat to achieve the same anti-pseudomonas effect and decrease microbialgrowth by 4 log, MBC would have been at best halved when the extractsare combined as compared to MBC when extracts were applied alone.Surprisingly, here, MBC were decreased by 2.5-5 times. This is synergy.

Still further unexpectedly, the anti-clostridium effect of thecombination of rosemary and Punica extracts, exceeded their expectedadditional effects when extracts were applied alone. Indeed, it wouldhave been expected that to achieve the same anti-clostridium effect anddecrease microbial growth by 4 log, MIB would have been at best halvedwhen the extracts are combined as compared to MIB when extracts wereapplied alone. This is synergy. Note, Punica extract had a MIC effect of0.016%. This implies that its MBC must be higher than MIC, meaninghigher than 0.016%. Either way, it is further unexpected that the MBC ofthe combination of rosemary and Punica extracts is more than twice lowerthan the MIC of an extract alone.

TABLE 20 Unexpected/Synergistic MIC effects of R and P (% in testsolution) Expected Measured MIC MIC MIC MIC of R (%) of P (%) of R/P (%)of R/P (%) S. mutans 0.25 >1 0.25 0.0625* S. aureus 0.125 0.50.125-0.325 0.0625* C. albicans 1 >1 1 0.25* S. cerevisiae 0.25 >1 0.250.0625*

Unexpectedly, when applied in combination, the minimal concentration ofthe combination R/P to inhibit microbial growth of bacteria or of yeast,S. mutans, S. aureus, C. albicans and S. cerevisiae is surprisingly low;it is 2-4 times lower than expected. Surprisingly, when the two extractsare combined, their activity is synergistically increased by 2.5-5 timesthan expected.

Further unexpectedly, the anti-streptococcus effect of the combinationof rosemary and Punica extracts, exceeded their expected additionaleffects when extracts were applied alone. Indeed, it would have beenexpected that to achieve the same anti-streptococcus effect and inhibitmicrobial growth, MIC would have been at best at the lowest MIC ascompared to MIC when extracts were applied alone. Surprisingly, here,MIC were decreased by 4 times than expected. This is synergy.

Still further unexpectedly, the anti-staphylococcus effect of thecombination of rosemary and Punica extracts, exceeded their expectedadditional effects when extracts were applied alone. Indeed, it wouldhave been expected that to achieve the same anti-staphylococcus effectand inhibit microbial growth, MIC would have been at best halved whenthe extracts are combined as compared to MIC when extracts were appliedalone. Surprisingly, here, MIC were decreased by 2-4.5 times thanexpected. This is synergy.

Further unexpectedly, the anti-candida effect of the combination ofrosemary and Punica extracts, exceeded their expected additional effectswhen extracts were applied alone. Indeed, it would have been expectedthat to achieve the same anti-candida effect and inhibit microbialgrowth, MIC would have been at best at the lowest MIC as compared to MICwhen extracts were applied alone. Surprisingly, here, MIC were decreasedby 4 times than expected. This is synergy.

And, still further unexpectedly, the anti-saccharomyces effect of thecombination of rosemary and Punica extracts, exceeded their expectedadditional effects when extracts were applied alone. Indeed, it wouldhave been expected that to achieve the same anti-saccharomyces effectand inhibit microbial growth, MIC would have been at best at the lowestMIC as compared to MIC when extracts were applied alone. Surprisingly,here, MIC were decreased by 4 times than expected. This is synergy.

It is noted that P. aeruginosa and S. typhimirium are Gram negativebacteria. C. perfringens, S. mutans, S. aureus and L. monocytogenes areGram positive bacteria. C. albicans and S. cerevisiae are yeast.

Rosemary in combination with Punica extract clearly exhibits synergisticantimicrobial effects against the growth of Gram positive (including L.monocytogenes (in meat), C. perfringens, S. mutans, S. aureus), Gramnegative (including S. typhimirium, P. aeruginosa) bacteria and yeast(including C. albicans, Saccharomyces cerevisiae).

TABLE 21 Unexpected/Synergistic effects of the combination of extractsR/P on different microorganisms are here summarized Nature of GramAntimicrobial Microorganism microorganism (+ or −) effect L.monocytogenes Bacteria G+ Unexpected, Synergy S. typhimirium Bacteria G−Unexpected, Synergy P. aeruginosa Bacteria G− Unexpected, Synergy C.perfringens Bacteria G+ Unexpected, Synergy S. mutans Bacteria G+Unexpected, Synergy S. aureus Bacteria G+ Unexpected, Synergy C.albicans Yeast Unexpected, Synergy S. cerevisiae Yeast Unexpected,Synergy

Note that hesperidin in combination with rosemary had no bactericidaleffect in vitro on Listeria monocytogenes even at highest here testedconcentrations: 2.5%.

Yet, surprisingly, such extract combination significantly andefficiently inhibited the growth of Listeria monocytogenes in meatproducts in synergistic manner.

Similar observation could be drawn when comparing hesperidin effects invitro, as reported in prior art against Listeria monocytogenes growthand in meat (this study). Indeed, although hesperidin in vitro did nothave antilisterial effects, it inhibited listerial growth in beef meat.

Use of Rosemary/Punica and Rosemary/Hesperidin Extracts inMeat/Poultry/Fish Matrices

Processing Methods, Extract Incorporation, Listerial Contamination andGrowth Inhibition

Fresh pork sausages and poultry were produced according to standardrecipes used by industry. As far as fish and seafood are concerned, hereexampled by smoked salmon, its processing was also done according to theindustrial procedures.

Meat/Poultry/Fish Batches

Whenever possible, to overcome the variability of raw materials in termsof pH and endogenous flora (rate and nature of the constituent flora)for each meat/category, the processing was carried out on 3 batches ofraw materials originating from different meat/poultry/fishsuppliers/(salmon) farms. Plant extracts or their combinations wereincorporated at the beginning of each recipe, together with basicprocessing ingredients. The plant extracts or their combinations wereincorporated in food matrices at 1-3 different concentrations, asexplained in Tables 23, 24, 26, 27, 29, and 30 below.

As far as every product category is concerned, controls, i.e. productsnot comprising plant extracts, were tested.

Nature and Origin of Strains of Listeria monocytogenes/BacterialPreparation Conditions

Two strains of Listeria monocytogenes in mixture (50/50) were studiedaccording to the Standard Operating Procedure NF V01-009 and concerningany meat/poultry/fish product. As far as tests in meat products areconcerned: the reference strain CIP 7838 (serovar 4b) and a so-calledfield strain isolated from pork (ADIV collection) serotype 1/2a, the onethat predominates in more than 50% on the fresh pork were used.

As far as poultry meat is concerned, the reference strain 7838 wascoupled to a strain of Listeria monocytogenes isolated from poultrycarcass (ADIV collection; serotype 1/2b). As far as tests in salmon areconcerned: the reference strain CIP 7838 was mixed with a strainisolated from salmon that ADIV was donated by a partner of its network.As far as any here studied food matrix is concerned, the two strainswere prepared and inoculated according to the guidelines of StandardOperating Procedures NF V01-009 (Version 2014).

Frozen strains, preserved as cryobilles (−80° C.), were revived andcultivated individually. Each strain was revived by transplanting 0.1mlin 10 ml of BHI culture medium (for 24 h at 30° C.). Two successivesubcultures have resulted in a pre-culture of each strain. Subsequently,each strain was again cultured for 24 hours at 30° C. The latter culturewas used in matrix product contamination (at end of the exponentialphase or early stationary phase). After two successive centrifugations,the pellets were suspended in 10 ml of buffered peptone water (BPW),counts were performed on BHI medium and the bacterial solution was keptat 0° C. for 24 hours prior to inoculation. After reading of thebacterial growth, the concentrations were then adjusted so as toinoculate the food matrix product with the mixture of the two strains(50/50) at 2-3 log cfu/g.

Inoculation Method

As specified in the NF V01-009, the products inoculation mode shouldmatch the reality of industrial contamination. Thus, sausages (pork orpoultry) were infected by inoculation in the mass of the meat mix tosimulate contamination from meat As the contamination of smoked salmonoccurs during the handling of raw materials or when slicing/packaging,here contamination of the surface of salmon slices was applied.

The inoculation method on the surface of the products has been developedby ADIV and provided an accurate weight of the inoculum, calculated in away to obtain the desired concentration at the surface of the products.Irrespectively of the method of inoculation (ground or surface), inorder to maintain the adequate water activity of food products, theaqueous volume to be added to the food matrix was calculated so as notto exceed a weight/volume ratio of 1/100 (NF V01-009).

Meat/Poultry/Fish Product Elaboration and Extract Incorporation

Pork Sausages

Pork sausages were manufactured according to a conventional process, incompliance with the code of practice, from lean pork (86%) and pork fat(14%). The initial meat mix intended for sausage manufacturing wasobtained by grinding/mincing fat and lean parts of pork meat atlow-temperature trough a grid (6 mm grid). Then, here tested plantextracts were added whenever applicable, according to Table 24 and Table25. After homogenization, the mix was then stuffed into natural casings(sheep menus, 24/26 diameter).

Contamination/Packaging

For a given batch, a meat mix of 42 kg was prepared and then dividedinto different sets of 5 kg each for the production of test series. Five(5) kg of meat mix were added to a mix prepared that did not contain anyantimicrobial agent nor additive (Mix Fraiche 230 South at 23 g/kg) and50 g water/kg. After homogenization, thus obtained mixture was dividedinto parts. A portion without artificial listerial contamination, wasplaced in trays (6 sausages per tray) that were packaged in MAPconditions (70% O₂/30% CO₂) with a conservation time scenario ⅓ ofconservation time at 4° C. and ⅔ of conservation time at 8° C. The otherpart was artificially contaminated by Listeria monocytogenes at anaverage rate of 3 log cfu/g (inoculation in the ground meat mass) beforebeing packaged and conserved as described above. The contaminated meatsamples that did not comprise plant extracts were termed control.

Analytical Monitoring During the Conservation

Microbiological analyses were performed on D0 and JDLC (D14) and theyconcerned the enumeration of Listeria monocytogenes, the totalmesophilic acidifying flora (FAM) and the lactic flora. At each point ofanalysis, a single repeat batch was performed by analysis of a tray (n=3for the 3 batches).

Manufacturing of Poultry Sausages

Poultry sausages were produced according to the same experimentalconditions as described for pork sausages. The only difference remainsin the nature of the raw materials used and the nature of compounds thatmay have been added. Indeed, in this case, the lean was made using upperparts of chicken thighs 85% and fat represented by the skin.

The ground meat mix did not include any classical antimicrobial butcontained a mix from which any ingredients having antimicrobial orantioxidative activity were removed (Mix Chipo Flight 310 to 31 g/kg).Thus prepared mixture either comprised plant extracts, or not (controls)and followed the same experimental conditions and listerial/bacterialanalyses as described for pork.

Preparation and Inoculation of Smoked Salmon

The manufacture of smoked salmon was conducted according to classicalprocedures well known from and available to a person skilled in the art.As for meat products, three different batches of salmon from threedifferent origins were made during the same week. The products werepackaged sliced on trays under vacuum (about 200 g per tray) on theindustrial site prior to listerial/bacterial inoculation and analysis.

Tested seafood, i.e. smoked salmon, were incorporated extractcombinations of Rosemary and hesperidin extracts, and of Rosemary andPunica extracts according to proportions and concentrations described inTables 29 and 30. The salmon was weighed and then surface-inoculated byspraying, with the mixture of the two strains (50/50) at an average rateof 3±0.5 log/g. After inoculation, the plates were again vacuum packedand put into storage at 8° C. for 30 days. Listerial growth wasmonitored upon inoculation (day 0) and on the 30^(th) day of growth.Non-inoculated controls were kept at cold storage for allmeet/poultry/fish matrices tested here for comparison purposes.

pH, total mesophilic acidifying flora, lactic flora were measuredthroughout all experiments in beef, pork, poultry meat or fish. Thepresence of here tested botanical extracts in these food matrices had nosignificant impact on pH, total mesophilic acidifying flora, lacticflora.

EXAMPLE Antimicrobial Effects of R/P and R/H in Poultry Sausages

Classical method, available to persons skilled in the art and based onthe protocol described for minced beef and above, was applied toprocessed poultry sausages.

As described above, briefly, three different batches of fresh poultrymeet processed into sausages, comprising R/P and R/H extractcombinations at different concentrations were inoculated with Listeriamonocytogenes. Controls did not comprise plant extracts. Fresh poultrymeat samples were kept in cool conditions and Listerial growth wasmeasured on the 14^(th) day.

TABLE 22 Initial Listerial load per batch of poultry sausages Batch L.monocytogenes (cfu/g) Batch 1 1.95E+03 Batch 2 2.93E+03 Batch 3 2.75E+03

Extracts were prepared as follows: Half quantities of R (0.5 R) and halfquantities of H (0.5H) or half quantities of P (0.5P) extracts asdefined above, were mixed together according to the Table 21 and, themix was completed up to 100% by maltodextrin. These powdered mixes wereadded to the poultry fresh meat upon processing into sausages inproportions as described in Tables 23 and 24. Tables 23 and 24 alsoindicate final contents in extracts and extract compounds in % and inppm, in extract and in the meat matrix.

Note that 0.5% of 0.5R+0.5H would correspond to the quantities inextract compounds termed 0.5R+0.5H that were tested in minced beef.Further note that 0.3% of 0.5R+0.5P would correspond to the quantitiesin extract compounds termed 0.5R+0.5P that were tested in minced beef.All obtained data were analyzed and expressed (i.e. delta log etc)according to explanations communicated regarding minced beef.

TABLE 23 Control 0.5R + 0.5H Composition Rosemary extract 0.00 3.05 ofextracts Carnosic acid 0.00 1.34 (%) Carnosic acid + carnosol 0.00 1.48Hesperidin extract 0.00 56.70 Hesperidin 0.00 53.87 0.6% CompositionRosemary extract 0 183 in poultry Carnosic acid 0 80 (ppm) Carnosicacid + carnosol 0 89 Hesperidin extract 0 3402 Hesperidin 0 3232

TABLE 24 Control LM 0.5R + 0.5P Composition Rosemary extract 0.00 5.33of extracts Carnosic acid 0.00 2.35 (%) Carnosic acid + carnosol 0.002.58 Pomegranate extract 0.00 21.60 Ellagic acid 0.00 0.43 Punicalagins0.00 1.94 0.2% 0.4% Composition Rosemary extract 0 107 213 in poultryCarnosic acid 0 47 94 (ppm) Carnosic acid + carnosol 0 52 103Pomegranate extract 0 432 864 Ellagic acid 0 9 17 Punicalagins 0 39 78

Results:

L. monocytogenes has clearly grown in control meat (FIG. 12.). Whencombinations of extracts were added to the meat, they efficientlyinhibited Listerial growth (FIG. 13 and FIG. 14). Those data confirm thedata found in minced beef which clearly demonstrated synergisticantilisterial effect of rosemary and hesperidin, and of rosemary andPunica extracts.

EXAMPLE Antimicrobial Effects of R/P and R/H in Pork Sausages

Classical method, available to people of ordinary skill in the art andbased on the protocol described for minced beef and above, was appliedto processed pork sausages.

Briefly, three different batches of fresh pork meet processed intosausages, comprising R/P and R/H extract combinations at differentconcentrations were inoculated with Listeria monocytogenes. Controls didnot comprise plant extracts. Fresh pork meat samples were kept in coolconditions (8° C.) and Listerial growth was measured on the 14^(th) day.

TABLE 25 Initial Listerial load per batch Batch L. monocytogenes (cfu/g)Batch 1 1.55E+03 Batch 2 2.96E+03 Batch 3 2.36E+03

Extracts were prepared as follows: Half quantities of R (0.5 R) and halfquantities of H (0.5H) or half quantities of P (0.5P) extracts asdefined above, were mixed together according to the Table 24 and, themix was completed up to 100% by maltodextrin. These powdered mixes wereadded to the pork fresh meat upon processing into sausages inproportions as described in Tables 26 and 27. Tables 26 and 27 alsoindicate final contents in extracts and extract compounds in % and inppm, in extract and in the meat matrix.

Note that 0.5% of 0.5R+0.5H would correspond to the quantities inextract compounds termed 0.5R+0.5H that were tested in minced beef. Alsonote that 0.3% of 0.5R+0.5P would correspond to the quantities inextract compounds termed 0.5R+0.5P that were tested in minced beef. Allobtained data were analyzed and expressed (i.e. delta log etc) accordingto explanations given above regarding minced beef.

TABLE 26 Control 0.5R + 0.5H Composition Rosemary extract 0.00 3.05 ofextracts Carnosic acid 0.00 1.34 (%) Carnosic acid + carnosol 0.00 1.48Hesperidin extract 0.00 56.70 Hesperidin 0.00 53.87 Control 0.4% 0.5%0.6% Composition Rosemary extract 0 122 153 183 in pork Carnosic acid 054 67 80 (ppm) Carnosic acid + carnosol 0 59 74 89 Hesperidin extract 02268 2835 3402 Hesperidin 0 2155 2694 3232 Control 0.5R + 0.5HComposition Rosemary extract 0.00 3.05 of extracts Carnosic acid 0.001.34 (%) Carnosic acid + carnosol 0.00 1.48 Hesperidin extract 0.0056.70 Hesperidin 0.00 53.87 0.4% 0.5% 0.6% Composition Rosemary extract0 122 153 183 in pork Carnosic acid 0 54 67 80 (ppm) Carnosic acid +carnosol 0 59 74 89 Hesperidin extract 0 2268 2835 3402 Hesperidin 02155 2694 3232

TABLE 27 Control LM 0.5R + 0.5P Composition Rosemary 0.00 5.33 ofextracts extract (%) Carnosic acid 0.00 2.35 Carnosic 0.00 2.58 acid +carnosol Pomegranate 0.00 21.60 extract Ellagic acid 0.00 0.43Punicalagins 0.00 1.94 0.2% 0.3% 0.4% Composition Rosemary 0 107 160 213in pork extract (ppm) Carnosic acid 0 47 71 94 Carnosic 0 52 77 103acid + carnosol Pomegranate 0 432 648 864 extract Ellagic acid 0 9 13 17Punicalagins 0 39 58 78

Results:

L. monocytogenes has clearly grown in control meat (FIG. 15). Whencombinations of extracts were added to the meat, they efficientlyinhibited Listerial growth at all here tested concentrations (FIG. 16and FIG. 17). Those data confirm the data found in minced beef whichclearly demonstrated a synergistic antilisterial effect of rosemary andhesperidin, and of rosemary and Punica extracts.

Classical method, available to persons skilled in the art and based onthe protocol described for minced beef and above, was applied to smokedsalmons.

As described above, briefly, three different batches of smoked salmon,comprising R/P and R/H extract combinations at different concentrationswere inoculated with Listeria monocytogenes. Controls did not compriseplant extracts. Smoked salmon samples were kept in cool conditions andListerial growth was measured on the 14^(th) day.

Initial Listerial load per batch of poultry sausages was as presented inTable 28:

TABLE 28 Initial Listerial load in smoked salmon Batch L. monocytogenes(cfu/g) Batch 1 1.46E+03 Batch 2 4.93E+03 Batch 3 5.64E+03

TABLE 29 Control 0.5R + 0.5H Composition Rosemary extract 0.00 3.05 ofextracts Carnosic acid 0.00 1.34 (%) Carnosic acid + carnosol 0.00 1.48Hesperidin extract 0.00 56.70 Hesperidin 0.00 53.87 Control 0.4% 0.5%Composition Rosemary extract 0 122 153 in smoked Carnosic acid 0 54 67salmon Carnosic acid + carnosol 0 59 74 (ppm) Hesperidin extract 0 22682835 Hesperidin 0 2155 2694

TABLE 30 Control LM 0.5R + 0.5P Composition Rosemary extract 0.00 5.33of extracts Carnosic acid 0.00 2.35 (%) Carnosic acid + carnosol 0.002.58 Pomegranate extract 0.00 21.60 Ellagic acid 0.00 0.43 Punicalagins0.00 1.94 0.3% Composition Rosemary extract 0 160 in smoked Carnosicacid 0 71 salmon Carnosic acid + carnosol 0 77 (ppm) Pomegranate extract0 648 Ellagic acid 0 13 Punicalagins 0 58

Note that 0.3% of 0.5R+0.5P corresponds to the same proportions anddosage of extract combination added in minced beef and termed 0.5R+0.5P.

Results:

L. monocytogenes has clearly grown in control smoked salmon (FIG. 18).When combinations of extracts were added to the meat, they efficientlyinhibited Listerial growth (FIG. 19 and FIG. 20). Those data confirm thedata found in minced beef which clearly demonstrated a synergisticantilisterial effect of rosemary and hesperidin, and of rosemary andPunica extracts.

REFERENCES

Numerous references have been cited throughout this disclosure. Allreferences cited in this disclosure including the three additionalreferences listed below are incorporated by reference.

Kai Reineke, Henning Weich, Dietrich Knorr, “The Influence of Sugars onPressure Induced Starch Gelatinization”, Procedia Food Science, Vol. 1,(2011), pages 2040-2046.

Shivangi Kelkar, Scott Stella, Carol Boushey, Martin Okos, “Developingnovel 3D measurement techniques and prediction method for food densitydetermination”, Procedia Food Science, Vol. 1, (2011), pages 483-491.

S. Chaillou, S.Christieans, M. Rivollier, I. Lucquin, M. C.Champomier-Vergès, M. Zagorec; “Quantification and efficiency ofLactobacillus sakei strain mixtures used as protective cultures inground beef”; Meat Science 97, (3) (2014), pages 332-338.

1-38. (canceled)
 39. An antimicrobial composition comprising aneffective amount of a phenolic diterpene and of hesperidin.
 40. Anantibacterial composition comprising an effective amount of a phenolicditerpene and of hesperidin.
 41. The composition according to claim 40wherein the composition is effective against Gram positive bacteriaselected from the group consisting of Bacillus cereus, Staphylococcusaureus, Streptococcus mutans, Listeria monocytogenes, Clostridiumperfringens, Enterococcus hirae and Mycobacterium bovis.
 42. Thecomposition according to claim 40 wherein the composition is effectiveagainst Gram negative bacteria selected from the group consisting ofPseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium andEnterobacter cloacae.
 43. An anti-fungal/anti-yeast compositioncomprising an effective amount of a phenolic diterpene and ofhesperidin.
 44. The composition according to claim 43 wherein thecomposition is effective against yeast selected from the groupconsisting of Saccharomyces cerevisiae and Candida albicans.
 45. Theantimicrobial composition according to claim 39 comprising purehesperidin and a Lamiaceae extract, wherein a majority of volatile oilcomponents from the lamiaceae extract having been removed.
 46. Theantibacterial composition according to claim 40 comprising purehesperidin and a Lamiaceae extract, wherein a majority of volatile oilcomponents from the lamiaceae extract having been removed.
 47. Theantimicrobial composition according to claim 45, wherein the purehesperidin contains from 80% to 99% hesperidin.
 48. The antibacterialcomposition according to claim 46, wherein the pure hesperidin containsfrom 80% to 99% hesperidin.
 49. The anti-fungal/anti-yeast compositionaccording to claim 43 comprising pure hesperidin and a Lamiaceaeextract, wherein a majority of volatile oil components from thelamiaceae extract having been removed.
 50. The anti-fungal/anti-yeastcomposition according to claim 49, wherein the pure hesperidin containsfrom 80% to 99% hesperidin.
 51. A food product comprising a food and thecomposition of claim
 39. 52. A food product comprising a food and thecomposition of claim
 40. 53. The food product according to claim 51,wherein the food is selected from the group consisting of fresh meat,fish and poultry.
 54. The food product according to claim 52, whereinthe food is selected from the group consisting of fresh meat, fish andpoultry.